Antibodies binding AXL

ABSTRACT

The present invention relates to anti-AXL antibodies, immunoconjugates, compositions and method of treatment of cancer with such anti-AXL antibodies, immunoconjugates, or compositions.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/325,364, filed Jan. 10, 2017, which is a 35 U.S.C. 371 national stagefiling of International Application No. PCT/EP2015/065900, filed Jul.10, 2015, which claims priority to Danish Patent Application Nos. PA2015 00283, PA 2014 00746, PA 2014 00489 and PA 2014 00380, filed on May12, 2015, Dec. 22, 2014, Sep. 1, 2014, and Jul. 11, 2014, respectively.The contents of the aforementioned applications are hereby incorporatedby reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Feb. 7, 2019, is namedGMI_161CUSDV_Sequence_Listing.txt and is 114,062 bytes in size.

FIELD OF INVENTION

The present invention relates to antibodies binding AXL,immunoconjugates, compositions comprising such antibodies orimmunoconjugates, and uses of said antibodies and immunoconjugates.

BACKGROUND

The TAM subfamily of mammalian Receptor Tyrosine Kinases (RTKs) consistsof AXL, Tyro3 and Mer. AXL is a 104-140 kDa transmembrane protein whichhas transforming abilities [1]. AXL can be activated upon binding of itsligand, the vitamin K-dependent growth arrest-specific factor 6 (Gas6).Gas6 binding to AXL leads to AXL dimerization, autophosphorylation andsubsequent activation of intracellular signaling pathways, such as thePI3K/AKT, mitogen-activated protein kinase (MAPK), STAT and NF-κBcascades [2]. In cancer cells, AXL enhances tumor cell motility,invasion, migration, and is involved in epithelial-to-mesenchymaltransition (EMT) [3]. Furthermore, AXL expression has been implicated inresistance to chemotherapy and targeted therapy, such as EpidermalGrowth Factor Recptor (EGFR) targeted therapy (Wilson 2014, Brand 2013,Zhang 2012) or inhibitors of the B-raf (BRAF) pathway (Muller, 2014).

The extracellular domain of TAM receptor family members is composed of acombination of two N-terminal immunoglobulin (Ig)-like domains and twofibronectin Type III (FNIII) repeats [1]. The ligand Gas6 binds to theIg-like domains I and II of AXL [14].

Upregulation of AXL has been reported in a variety of cancers, includinggastric, prostate, ovarian, and lung cancer [1]. Furthermore, AXL isoverexpressed in breast and pancreatic cancers and is significantlyassociated with higher metastasis frequency and with poor overallsurvival [2].

Targeted inhibition of RTKs may be effective as anti-tumor and/ormetastatic therapy. Such targeted inhibition of AXL and/or the ligandGas6 comprises both small molecules and anti-AXL antibodies [3].Anti-AXL antibodies have been described that attenuate non-small celllung carcinoma xenograft growth in vivo by downregulation of receptorexpression, reducing tumor cell proliferation and inducing apoptosis[4]. Furthermore, various monoclonal antibodies have been described thatblock binding of the ligand Gas6 to AXL [2], [5], and [7].

Anti-AXL antibodies have been described previously [8]-[13]. However, aneed for anti-AXL antibodies having improved anti-tumor activityremains.

SUMMARY OF INVENTION

It is an object of the present invention to provide anti-AXL antibodies.Thus, in one aspect, the present invention relates to an antibody whichbinds to AXL, wherein the antibody, does not compete for AXL bindingwith the ligand Growth Arrest-Specific 6 (Gash).

In another aspect, the present invention relates to a bispecificantibody comprising a first binding region of an antibody according tothe invention, and a second binding region which binds a differenttarget or epitope than said first antigen-binding region.

In another aspect, the present invention relates to an immunoconjugatecomprising the antibody or bispecific antibody according to theinvention, and a therapeutic moiety, such as a cytotoxic agent, achemotherapeutic drug, a cytokine, an immunosuppressant, antibiotic, ora radioisotope.

In another aspect, the present invention relates to a compositioncomprising the antibody, bispecific antibody, or immunoconjugateaccording to the invention.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising the antibody, bispecific antibody, orimmunoconjugate according to the invention, and a pharmaceuticallyacceptable carrier.

In another aspect, the present invention relates to a nucleic acidconstruct encoding an antibody according to the invention.

In another aspect, the present invention relates to an expression vectorcomprising one or more nucleic acid constructs according to theinvention.

In another aspect, the present invention relates to a host cellcomprising a vector according to the invention.

In another aspect, the present invention relates to a hybridoma whichproduces the antibody according to the invention.

In another aspect, the present invention relates to the antibody,bispecific antibody, or immunoconjugate according to the invention foruse as a medicament.

In another aspect, the present invention relates to the antibody,bispecific antibody, or immunoconjugate according to the invention foruse in the treatment of cancer.

In another aspect, the present invention relates to a method oftreatment of cancer comprising administering the antibody, bispecificantibody, immunoconjugate, composition, or pharmaceutical compositionaccording to the invention, to a subject in need thereof.

In another aspect, the present invention relates to a method ofdiagnosing a disease characterized by involvement or accumulation ofAXL-expressing cells, comprising administering an antibody, bispecificantibody, immunoconjugate, composition, or a pharmaceutical compositionaccording to the invention, to a subject, optionally wherein theantibody is labeled with a detectable agent, and wherein the amount ofAXL-expressing cells correlates with or is indicative of disease.

In another aspect, the present invention relates to a method forinhibiting growth and/or proliferation of a tumor cell expressing AXL,comprising administration, to an individual in need thereof, of anantibody, bispecific antibody, immunoconjugate, composition, orpharmaceutical composition according to the invention.

In another aspect, the present invention relates to a method forproducing an antibody according to the invention, the method comprisingthe steps a) culturing a host cell or hybridoma according to theinvention, and b) purifying the antibody from the culture media.

In another aspect, the present invention relates to a diagnosticcomposition comprising an antibody or bispecific antibody according tothe invention.

In another aspect, the present invention relates to a method fordetecting the presence of AXL antibody, or a cell expressing AXL, in asample comprising the steps of a) contacting the sample with anantibody, bispecific antibody, immunoconjugate according to theinvention, under conditions that allow for formation of a complexbetween the antibody, bispecific antibody, or immunoconjugate and AXL;and b) analyzing whether a complex has been formed.

In another aspect, the present invention relates to a kit for detectingthe presence of AXL antigen, or a cell expressing AXL, in a samplecomprising i) an antibody, bispecific antibody, or immunoconjugateaccording to the invention; and ii) instructions for use of the kit.

In another aspect, the present invention relates to an anti-idiotypicantibody which binds to an anti-AXL antibody according to the invention.s

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-1C: Binding curves of anti-AXL antibodies to HEK293 cellstransfected with (FIG. 1A) human AXL-ECD, (FIG. 1B) cynomolgus AXL-ECD,or (FIG. 1C) mouse AXL-ECD. Data shown are mean fluorescence intensities(MFI) of one representative experiment, as described in Example 2.

FIGS. 2A-2F: Binding of anti-AXL antibodies to mouse-human AXL chimeraswas performed as described in Example 3. The following Homo sapiens AXL(hsAXL) and Mus musculus AXL (mmAXL) chimeric proteins were tested:(FIG. 2A) hsAXL and mock, (FIG. 2B) hsAXL-mmECD, (FIG. 2C) hsAXL-mmIg1,(FIG. 2D) hsAXL-mmIg2, (FIG. 2E) hsAXL-mmFN1, (FIG. 2F) hsAXL-mmFN2.

FIG. 3: Anti-AXL antibody-dependent cell-mediated cytotoxicity in A431cells. Antibody-dependent cell-mediated cytotoxicity by anti-AXLantibodies in A431 cells was determined as described in Example 4.

FIG. 4: Binding characteristics of AXL antibody-drug conjugates(AXL-ADCs). Binding of AXL-ADCs on HEK293T cells transiently transfectedwith human AXL was determined as described in Example 5. Data shown aremean fluorescence intensities (MFI) of one representative experiment.

FIGS. 5A and 5B: In vitro cytotoxicity induced by AXL antibody-drugconjugates. Induction of cytotoxicity by AXL antibody-drug conjugateswas determined as explained in Example 6.

FIGS. 6A-6E: Antibody VH and VL variants that allow binding to AXL.Antibodies with identical VL or VH regions were aligned and differencesin VH (FIGS. 6A-6D) or VL (FIG. 6E) sequences, respectively, wereidentified and indicated by boxes in the figures. CDR regions areunderlined.

FIG. 7: Induction of cytotoxicity by ADCs in LCLC-103H cells wasdetermined as described in Example 8.

FIG. 8: Anti-tumor activity by MMAE-conjugated AXL antibodies in atherapeutic LCLC-103H xenograft model as described in Example 9.

FIG. 9: Immunohistochemical staining of frozen PAXF1657 tumor sections(pancreas cancer PDX model) using a pool of AXL monoclonal antibodies asdescribed in Example 10.

FIGS. 10A-10C: (FIG. 10A) Average tumor size after therapeutic treatmentwith AXL-ADCs the PAXF1657 model. An unconjugated AXL Humab (FIG. 10B)and an untargeted ADC (FIG. 10C) do not show anti-tumor activity,indicating that the therapeutic capacity of AXL-ADCs was dependent onthe cytotoxic activity of MMAE and on target binding, error barsrepresent S.E.M.

FIGS. 11A-11F: Binding of anti-AXL antibodies to mouse-human AXLchimeras was performed as described in Example 11. The following Homosapiens AXL (hsAXL) and Mus musculus AXL (mmAXL) chimeric proteins weretested: (FIG. 11A) hsAXL and mock, (FIG. 11B) hsAXL-mmECD, (FIG. 11C)hsAXL-mmIg1, (FIG. 11D) hsAXL-mmIg2, (FIG. 11E) hsAXL-mmFN1, (FIG. 11F)hsAXL-mmFN2.

FIG. 12: Binding of human Gas6 (hGas6) on A431 cells that had beenpre-incubated with antibodies binding to the Ig1 domain of AXL. Datashown are mean fluorescence intensities (MFI) of one representativeexperiment.

FIGS. 13A and 13B: Anti-tumor activity of MMAE-conjugated AXL antibodiesin a therapeutic A431 xenograft model, that produces high levels ofendogeneous Gas6, as described in Example 13. FIGS. 13A and 1313 showresults from 2 independent experiments.

FIGS. 14A and 14B: Anti-tumor activity of MMAE-conjugated AXL antibodiesin a therapeutic LCLC-103H xenograft model, that expresses low levels ofendogenous Gas6, as described in Example 13.

FIGS. 14A and 14B show results from 2 independent experiments.

FIGS. 15A and 15B: Induction of cytotoxicity by AXL-ADCs in A431 cells(FIG. 15A) and MDA-MB231 cells (FIG. 15B) was determined as described inExample 8.

FIG. 16. AXL staining in thyroid, esophageal, ovarian, breast, lung,pancreatic, cervical and endometrial cancer. The average AXL stainingintensity (OD) of AXL-positive cells is plotted on the X-axis, and thepercentage of AXL-positive tumor cells is plotted on the Y-axis. Eachdot represents a tumor core, derived from an individual patent.

FIG. 17. Representative examples of AXL-immunostained tumor cores fordifferent tumor indication.

FIGS. 18A-18G. AXL antibodies specifically bind AXL but not to other TAMreceptor family members. Binding of HuMab-AXL antibodies to HEK293 cellstransfected with human AXL (FIG. 18A), human MER (FIG. 18B), human TYRO3(FIG. 18C), or untransfected HEK293 cells (FIG. 18D). To confirm properexpression of transfected cells, untransfected HEK293F cells and cellstransfected with AXL (FIG. 18E), MER (FIG. 18F), or TYRO3 (FIG. 18G)were stained with MER- and TYRO3-specific antibodies. Data shown aremean fluorescence intensities (MFI) of one representative experiment, asdescribed in Example 15.

FIGS. 19A-19D. Detection of AXL antibodies on the plasma membrane oftumor cell lines that had been incubated with AXL-antibodies for 1 hourat 4° C., followed by an overnight incubation 4° C. or 37° C. In bothMDA-MB-231 (FIGS. 19A and 19B) and Calu-1 cells (FIGS. 19C and 19D),more antibody was detected on the plasma membrane of cells that had beenincubated at 4° C. than on cells that had been incubated at 37° C.,illustrating internalization of membrane-bound antibody at 37° C.

FIG. 20. Geomean fluorescence intensity of LCLC-103H cells afterincubation with AXL antibodies that had been complexed toFab-TAMRA/QSY7. IgG1-b12 and Fab-TAMRA/QSY7 alone were included asnegative controls.

FIGS. 21A and 21B. (FIG. 21A) Average tumor size after therapeutictreatment with IgG1-AXL-107-vcMMAE in the esophageal cancer PDX modelES0195. IgG1-b12 and IgG1-b12-MMAE were included as isotype controlantibody and isotype control ADC, respectively. (FIG. 21B) Tumor size inindividual mice on day 32 after injection of MDA-MB-231-luc D3H2LN tumorcells in the mammary fat pads of female SCID mice. * p<0.05; ** p<0.0001

FIGS. 22A and 22B. Therapeutic effect of AXL-ADCs in a patient-derivedcervical cancer xenograft model. (FIG. 22A) Average tumor size aftertherapeutic treatment with IgG1-AXL-183-vcMMAE or IgG1-AXL-726-vcMMAE inthe cervical cancer PDX model CEXF 773. IgG1-b12 and IgG1-b12-MMAE wereincluded as isotype control antibody and isotype control ADC,respectively. (FIG. 22B) Tumor size in individual mice on day 28 afterinitiation of treatment in the cervical cancer PDX model CEXF 773. *p<0.001.

FIGS. 23A and 23B. Therapeutic activity of AXL-ADCs in an orthotopicbreast cancer xenograft model. (FIG. 23A) Average tumor size aftertherapeutic treatment with IgG1-AXL-183-vcMMAE or IgG1-AXL-726-vcMMAE inan orthotopic MDA-MB-231-luc D3H2LN xenograft model. IgG1-b12 andIgG1-b12-MMAE were included as isotype control antibody and isotypecontrol ADC, respectively. (FIG. 23B) Tumor size in individual mice onday 32 after injection of MDA-MB-231-luc D3H2LN tumor cells in themammary fat pads of female SCID mice. * p<0.001.

FIG. 24. Cytotoxicity of IgG1-AXL-107-vcMMAE in human tumor cell lineswith different levels of AXL expression on the plasma membrane. AXLexpression in the plasma membrane of human tumor cell lines was assessedusing Qifikit analysis, and the cytotoxicity of IgG1-AXL-107-vcMMAE wasexpressed as the percentage of viable tumor cells that remained in thecell cultures after exposure to 1 μg/mL IgG1-AXL-107-vcMMAE.

DETAILED DESCRIPTION

Antibodies

In one aspect, the present invention relates to an antibody which bindsto AXL, wherein the antibody, does not compete for AXL binding with theligand Growth Arrest-Specific 6 (Gash).

The term “antibody” as used herein is intended to refer to animmunoglobulin molecule, a fragment of an immunoglobulin molecule, or aderivative of either thereof, which has the ability to specifically bindto an antigen under typical physiological and/or tumor-specificconditions with a half-life of significant periods of time, such as atleast about 30 minutes, at least about 45 minutes, at least about onehour, at least about two hours, at least about four hours, at leastabout 8 hours, at least about 12 hours, about 24 hours or more, about 48hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any otherrelevant functionally-defined period (such as a time sufficient toinduce, promote, enhance, and/or modulate a physiological responseassociated with antibody binding to the antigen and/or time sufficientfor the antibody to be internalized). The binding region (or bindingdomain which may be used herein, both having the same meaning) whichinteracts with an antigen, comprises variable regions of both the heavyand light chains of the immunoglobulin molecule. The constant regions ofthe antibodies (Abs) may mediate the binding of the immunoglobulin tohost tissues or factors, including various cells of the immune system(such as effector cells) and components of the complement system such asC1q, the first component in the classical pathway of complementactivation. As indicated above, the term antibody as used herein, unlessotherwise stated or clearly contradicted by context, includes fragmentsof an antibody that retain the ability to specifically interact, such asbind, to the antigen. It has been shown that the antigen-bindingfunction of an antibody may be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antibody” include (i) a Fab′ or Fab fragment, a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains, or a monovalent antibodyas described in [15]; (ii) F(ab′)₂ fragments, bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) an Fd fragment consisting essentially of the VH and CH1domains; (iv) an Fv fragment consisting essentially of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment [16], whichconsists essentially of a VH domain and is also called domain antibody[17]; (vi) camelid or nanobodies [18] and (vii) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they may be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainantibodies or single chain Fv (scFv), see for instance [19] and [20]).Such single chain antibodies are encompassed within the term antibodyunless otherwise noted or clearly indicated by context. Although suchfragments are generally included within the meaning of antibody, theycollectively and each independently are unique features of the presentinvention, exhibiting different biological properties and utility. Theseand other useful antibody fragments in the context of the presentinvention are discussed further herein. It also should be understoodthat the term antibody, unless specified otherwise, also includespolyclonal antibodies, monoclonal antibodies (mAbs), antibody-likepolypeptides, such as chimeric antibodies and humanized antibodies, aswell as ‘antibody fragments’ or ‘fragments thereof’ retaining theability to specifically bind to the antigen (antigen-binding fragments)provided by any known technique, such as enzymatic cleavage, peptidesynthesis, and recombinant techniques, and retaining the ability to beconjugated to a toxin. An antibody as generated can possess any isotype.

The term “immunoglobulin heavy chain” or “heavy chain of animmunoglobulin” as used herein is intended to refer to one of the heavychains of an immunoglobulin. A heavy chain is typically comprised of aheavy chain variable region (abbreviated herein as VH) and a heavy chainconstant region (abbreviated herein as CH) which defines the isotype ofthe immunoglobulin. The heavy chain constant region typically iscomprised of three domains, CH1, CH2, and CH3. The term “immunoglobulin”as used herein is intended to refer to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four potentially inter-connected by disulfide bonds. The structureof immunoglobulins has been well characterized (see for instance [21]).Within the structure of the immunoglobulin, the two heavy chains areinter-connected via disulfide bonds in the so-called “hinge region”.Equally to the heavy chains each light chain is typically comprised ofseveral regions; a light chain variable region (abbreviated herein asVL) and a light chain constant region. The light chain constant regiontypically is comprised of one domain, CL. Furthermore, the VH and VLregions may be further subdivided into regions of hypervariability (orhypervariable regions which may be hypervariable in sequence and/or formof structurally defined loops), also termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs). Each VH and VL is typically composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. CDR sequences are defined according to IMGT (see [22] and[23]).

The term “antigen-binding region” or “binding region” as used herein,refers to a region of an antibody which is capable of binding to theantigen. The antigen can be any molecule, such as a polypeptide, e.g.present on a cell, bacterium, or virion. The terms “antigen” and“target” may, unless contradicted by the context, be usedinterchangeably in the context of the present invention.

The term “binding” as used herein refers to the binding of an antibodyto a predetermined antigen or target, typically with a binding affinitycorresponding to a K_(D) of about 10⁻⁶ M or less, e.g. 10⁻⁷ M or less,such as about 10⁻⁸ M or less, such as about 10⁻⁹ M or less, about 10⁻¹⁰M or less, or about 10⁻¹¹ M or even less when determined by for instancesurface plasmon resonance (SPR) technology in a BIAcore 3000 instrumentusing the antigen as the ligand and the protein as the analyte, andbinds to the predetermined antigen with an affinity corresponding to aK_(D) that is at least ten-fold lower, such as at least 100 fold lower,for instance at least 1,000 fold lower, such as at least 10,000 foldlower, for instance at least 100,000 fold lower than its affinity forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The amount withwhich the affinity is lower is dependent on the K_(D) of the protein, sothat when the K_(D) of the protein is very low (that is, the protein ishighly specific), then the amount with which the affinity for theantigen is lower than the affinity for a non-specific antigen may be atleast 10,000 fold. The term “K_(D)” (M), as used herein, refers to thedissociation equilibrium constant of a particular antibody-antigeninteraction, and is obtained by dividing k_(d) by k_(a).

The term “k_(d)” (sec⁻¹), as used herein, refers to the dissociationrate constant of a particular antibody-antigen interaction. Said valueis also referred to as the k_(off) value or off-rate.

The term “k_(a)” (M⁻¹×sec⁻¹), as used herein, refers to the associationrate constant of a particular antibody-antigen interaction. Said valueis also referred to as the k_(on) value or on-rate.

The term “K_(A)” (M⁻¹), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing k_(a) by k_(d).

The term “AXL” as used herein, refers to the protein entitled AXL, whichis also referred to as UFO or JTK11, a 894 amino acid protein with amolecular weight of 104-140 kDa that is part of the subfamily ofmammalian TAM Receptor Tyrosine Kinases (RTKs). The molecular weight isvariable due to potential differences in glycosylation of the protein.The AXL protein consists of two extracellular immunoglobulin-like(Ig-like) domains on the N-terminal end of the protein, twomembrane-proximal extracellular fibronectin type III (FNIII) domains, atransmembrane domain and an intracellular kinase domain. AXL isactivated upon binding of its ligand Gas6, by ligand-independenthomophilic interactions between AXL extracellular domains, byautophosphorylation in presence of reactive oxygen species [24] or bytransactivation through EGFR (Meyer, 2013), and is aberrantly expressedin several tumor types. In humans, the AXL protein is encoded by anucleic acid sequence encoding the amino acid sequence shown in SEQ IDNO:130 (human AXL protein: Swissprot P30530; cynomolgus AXL protein:Genbank accession HB387229.1)).

The term “ligand-independent homophilic interactions” as used herein,refers to association between two AXL molecules (expressed onneighboring cells) that occurs in absence of the ligand.

The term “antibody binding AXL” as used herein, refers to any antibodybinding an epitope on the extracellular part of AXL.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids, sugar side chains or a combinationthereof and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. The epitope may comprise amino acid residues whichare directly involved in the binding, and other amino acid residues,which are not directly involved in the binding, such as amino acidresidues which are effectively blocked or covered by the specificantigen binding peptide (in other words, the amino acid residue iswithin the footprint of the specific antigen binding peptide).

The term “ligand” as used herein, refers to a substance, such as ahormone, peptide, ion, drug or protein, that binds specifically andreversibly to another protein, such as a receptor, to form a largercomplex. Ligand binding to a receptor may alter its chemicalconformation, and determines its functional state. For instance, aligand may function as agonist or antagonist.

The term “Growth Arrest-Specific 6” or “Gas6” as used herein, refers toa 721 amino acid protein, with a molecular weight of 75-80 kDa, thatfunctions as a ligand for the TAM family of receptors, including AXL.Gas6 is composed of an N-terminal region containing multiplegamma-carboxyglutamic acid residues (Gla), which are responsible for thespecific interaction with the negatively charged phospholipid membrane.Although the Gla domain is not necessary for binding of Gas6 to AXL, itis required for activation of AXL. Gas6 may also be termed as the“ligand to AXL”.

The terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibodycomposition”, “mAb”, or the like, as used herein refer to a preparationof antibody molecules of single molecular composition. A monoclonalantibody composition displays a single binding specificity and affinityfor a particular epitope. Accordingly, the term “human monoclonalantibody” refers to antibodies displaying a single binding specificitywhich have variable and constant regions derived from human germlineimmunoglobulin sequences. The human monoclonal antibodies may beproduced by a hybridoma which includes a B cell obtained from atransgenic or transchromosomal non-human animal, such as a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene, fused to an immortalized cell.

In one embodiment, maximal antibody binding in the presence of Gas6 isat least 90%, such as at least 95%, such as at least 97%, such as atleast 99%, such as 100%, of binding in absence of Gas6, as determined bythe method disclosed in Example 2.

Competition between anti-AXL and the ligand Gas6 to AXL may bedetermined as described in Example 2 under the heading “Interference ofanti-AXL binding with Gas6 binding”. Thus, in one embodiment, theantibody does not compete for AXL binding with the ligand Gas6, whereinthe competing for binding is determined in an assay comprising the stepsof

i) incubating AXL-expressing cells with Gas6,

ii) adding anti-AXL antibodies to be tested,

iii) adding a fluorescently labelled secondary reagent detectinganti-AXL antibodies and

iv) analyzing the cells by FACS.

In another embodiment, the antibody does not compete for binding withthe ligand Gas6, wherein the competing for binding is determined in anassay comprising the steps of

i) incubating AXL-expressing cells with anti-AXL antibodies,

ii) adding Gas6,

iii) adding a fluorescently labelled secondary reagent detecting Gas6,and

iv) analyzing the cells by FACS.

In one embodiment, the antibody has a binding affinity (K_(D)) in therange of 0.3×10⁻⁹ to 63×10⁻⁹ M to AXL, and wherein said binding affinityis measured using a Bio-layer Interferometry using soluble AXLextracellular domain.

The binding affinity may be determined as described in Example 2. Thus,in one embodiment, the antibody has a binding affinity of 0.3×10⁻⁹ to63×10⁻⁹ M to the antigen, wherein the binding affinity is determined bya method comprising the steps of;

i) loading anti-human Fc Capture biosensors with anti-AXL antibodies,and

ii) determining association and dissociation of soluble recombinant AXLextracellular domain by Bio-Layer Interferometry at differentconcentrations.

The term “soluble recombinant AXL extracellular domain” as used herein,refers to an AXL extracellular domain that has been expressedrecombinantly. Due to absence of the transmembrane and intracellulardomain, recombinant AXL extracellular domain is not attached to a, e.g.cell surface and stays in solution. It is well-known how to express aprotein recombinantly, see e.g. [25], and thus, it is within theknowledge of the skilled person to provide such recombinant AXLextracellular domain.

In one embodiment, the antibody has a dissociation rate of 6.9×10⁻⁵ s⁻¹to 9.7×10⁻³ s⁻¹ to AXL, and wherein the dissociation rate is measured byBio-layer Interferometry using soluble recombinant AXL extracellulardomain.

The binding affinity may be determined as described above (and inExample 2). Thus, in one embodiment, the antibody has a dissociationrate of 6.9×10⁻⁵ s⁻¹ to 9.7×10⁻³ s⁻¹ to AXL, and wherein thedissociation rate is measured by a method comprising the steps of

-   -   i) loading anti-human Fc Capture biosensors with anti-AXL        antibodies, and    -   ii) determining association and dissociation of recombinant AXL        extracellular domain by Bio-Layer Interferometry at different        concentrations.

The term “dissociation rate” as used herein, refers to the rate at whichan antigen-specific antibody bound to its antigen, dissociates from thatantigen, and is expressed as s⁻¹. Thus, in the context of an antibodybinding AXL, the term “dissociation rate”, refers to the antibodybinding AXL dissociates from the recombinant extracellular domain ofAXL, and is expressed as s⁻¹.

In one embodiment, AXL is human AXL. The amino acid sequence of AXL isaccording to Swissprot P30530.

In one embodiment, AXL is cynomolgus monkey AXL (Genbank accessionHB387229.1).

In one embodiment, the antibody comprises at least one binding regioncomprising variable heavy chain (VH) CDR1, CDR2, and CDR3 sequenceshaving at least 95%, such as at least 96%, such as at least 97%, such asat least 98%, such as at least 99%, sequence identity to sequences areselected from the group consisting of:

a) SEQ ID Nos.: 36, 37, and 38, respectively [107];

b) SEQ ID Nos.: 93, 94, and 95, respectively [613];

c) SEQ ID Nos.: 93, 126, and 127, respectively[613/608-01/610-01/620-06];

d) SEQ ID Nos.: 46, 47, and 48, respectively [148];

e) SEQ ID Nos.: 57, 58, and 59, respectively [171];

f) SEQ ID Nos.: 78, 79, and 80, respectively [187];

g) SEQ ID Nos.: 46, 119, and 120, respectively [148/140];

h) SEQ ID Nos.: 51, 52, and 53, respectively [154];

i) SEQ ID Nos.: 72, 73, and 75, respectively [183];

j) SEQ ID Nos.: 72, 74, and 75, respectively [183-N52Q];

k) SEQ ID Nos.: 114, 115, and 116, respectively [733];

l) SEQ ID Nos.: 123, 124, and 125, respectively [171/172/181];

m) SEQ ID Nos.: 108, 109, and 110, respectively [726];

n) SEQ ID Nos.: 108, 121, and 122, respectively [726/187];

o) SEQ ID Nos.: 41, 42, and 43, respectively [140];

p) SEQ ID Nos.: 62, 63, and 64, respectively [172];

q) SEQ ID Nos.: 67, 68, and 69, respectively [181];

r) SEQ ID Nos.: 51, 52, and 54, respectively [154-M103L];

s) SEQ ID Nos.:78, 79, and 80, respectively [187];

t) SEQ ID Nos.: 83, 84, and 85, respectively [608-01];

u) SEQ ID Nos.: 88, 89, and 90, respectively [610-01];

v) SEQ ID Nos.: 98, 99, and 100, respectively, [613-08];

w) SEQ ID Nos.: 103, 104, and 105, respectively [620-06]; and

x) SEQ ID Nos.: 108, 109, and 111, respectively [726-M101L].

In one embodiment, the antibody comprises at least one binding regioncomprising variable heavy chain (VH) CDR1, CDR2, and CDR3 sequenceshaving at most 5 mutations or substitutions, such as at most 4 mutationsor substitutions, such as at most 3 mutations or substitutions, such asat most 2 mutations or substitutions, such as at most 1 mutation orsubstitution, in total across the CDR sequences in said variable heavychain selected from the group consisting of:

a) SEQ ID Nos.: 36, 37, and 38, respectively [107];

b) SEQ ID Nos.: 93, 94, and 95, respectively [613];

c) SEQ ID Nos.: 93, 126, and 127, respectively[613/608-01/610-01/620-06];

d) SEQ ID Nos.: 46, 47, and 48, respectively [148];

e) SEQ ID Nos.: 57, 58, and 59, respectively [171];

f) SEQ ID Nos.: 78, 79, and 80, respectively [187];

g) SEQ ID Nos.: 46, 119, and 120, respectively [148/140];

h) SEQ ID Nos.: 51, 52, and 53, respectively [154];

i) SEQ ID Nos.: 72, 73, and 75, respectively [183];

j) SEQ ID Nos.: 72, 74, and 75, respectively [183-N52Q];

k) SEQ ID Nos.: 114, 115, and 116, respectively [733];

l) SEQ ID Nos.: 123, 124, and 125, respectively [171/172/181];

m) SEQ ID Nos.: 108, 109, and 110, respectively [726];

n) SEQ ID Nos.: 108, 121, and 122, respectively [726/187];

o) SEQ ID Nos.: 41, 42, and 43, respectively [140];

p) SEQ ID Nos.: 62, 63, and 64, respectively [172];

q) SEQ ID Nos.: 67, 68, and 69, respectively [181];

r) SEQ ID Nos.: 51, 52, and 54, respectively [154-M103L];

s) SEQ ID Nos.:78, 79, and 80, respectively [187];

t) SEQ ID Nos.: 83, 84, and 85, respectively [608-01];

u) SEQ ID Nos.: 88, 89, and 90, respectively [610-01];

v) SEQ ID Nos.: 98, 99, and 100, respectively, [613-08];

w) SEQ ID Nos.: 103, 104, and 105, respectively [620-06]; and

x) SEQ ID Nos.: 108, 109, and 111, respectively [726-M101L].

Hereby embodiments are provided wherein mutations or substitutions of upto five mutations or substitutions are allowed across the three CDRsequences in the variable heavy chain. The mutations or substitutionsmay be of conservative, physical or functional amino acids such thatmutations or substitutions do not change the epitope or preferably donot modify binding affinity to the epitope more than 30%, such as morethan 20% or such as more than 10%. The conservative, physical orfunctional amino acids are selected from the 20 natural amino acids i.e.Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile,Leu, Met, Phe, Trp, Tyr and Val.

In one embodiment, the antibody comprises at least one binding regioncomprising variable heavy chain (VH) CDR1, CDR2, and CDR3 sequences areselected from the group consisting of;

a) SEQ ID Nos.: 36, 37, and 38, respectively [107];

b) SEQ ID Nos.: 93, 94, and 95, respectively [613];

c) SEQ ID Nos.: 93, 126, and 127, respectively[613/608-01/610-01/620-06];

d) SEQ ID Nos.: 46, 47, and 48, respectively [148];

e) SEQ ID Nos.: 57, 58, and 59, respectively [171];

f) SEQ ID Nos.: 78, 79, and 80, respectively [187];

g) SEQ ID Nos.: 46, 119, and 120, respectively [148/140];

h) SEQ ID Nos.: 51, 52, and 53, respectively [154];

i) SEQ ID Nos.: 72, 73, and 75, respectively [183];

j) SEQ ID Nos.: 72, 74, and 75, respectively [183-N52Q];

k) SEQ ID Nos.: 114, 115, and 116, respectively [733];

l) SEQ ID Nos.: 123, 124, and 125, respectively [171/172/181];

m) SEQ ID Nos.: 108, 109, and 110, respectively [726];

n) SEQ ID Nos.: 108, 121, and 122, respectively [726/187];

o) SEQ ID Nos.: 41, 42, and 43, respectively [140];

p) SEQ ID Nos.: 62, 63, and 64, respectively [172];

q) SEQ ID Nos.: 67, 68, and 69, respectively [181];

r) SEQ ID Nos.: 51, 52, and 54, respectively [154-M103L];

s) SEQ ID Nos.:78, 79, and 80, respectively [187];

t) SEQ ID Nos.: 83, 84, and 85, respectively [608-01];

u) SEQ ID Nos.: 88, 89, and 90, respectively [610-01];

v) SEQ ID Nos.: 98, 99, and 100, respectively, [613-08];

w) SEQ ID Nos.: 103, 104, and 105, respectively [620-06]; and

x) SEQ ID Nos.: 108, 109, and 111, respectively [726-M101L].

In one particular embodiment, the VH CDR1, CDR2, and CDR3 are selectedfrom either a), d), g), or k).

In one embodiment, the at least one binding region comprises a VH regionand a variable light chain (VL) region having at least 95%, such as atleast 97%, such as at least 99%, such as 100%, sequence identity withthe sequences independently selected from the group consisting of;

a) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 36, 37, and 38, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 39, GAS, and 40, respectively,[107];

b) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 46, 47, and 48, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 49, AAS, and 50, respectively,[148];

c) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 114, 115, and 116, respectively, and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 117, DAS, and 118,respectively [733];

d) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 51, 52, and 53, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 55, GAS, and 56, respectively[154];

e) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 51, 52, and 54, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 55, GAS, and 56, respectively[154-M103L];

f) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 57, 58, and 59, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 60, GAS, and 61, respectively,[171];

g) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 62, 63, and 64, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 65, GAS, and 66, respectively,[172];

h) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 67, 68, and 69, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 70, GAS, and 71, respectively,[181];

i) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 72, 73, and 75, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 76, ATS, and 77, respectively,[183];

j) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 72, 74, and 75, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 76, ATS, and 77, respectively,[183-N52Q];

k) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 78, 79, and 80, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 81, AAS, and 82, respectively,[187];

l) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 83, 84, and 85, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 86, GAS, and 87, respectively,[608-01];

m) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 88, 89, and 90, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 91, GAS, and 92, respectively,[610-01];

n) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 93, 94, and 95, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 96, GAS, and 97, respectively,[613];

o) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 98, 99, and 100, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 10, DAS, and 102,respectively, [613-08];

p) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 103, 104, and 105, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 106, GAS, and 107,respectively, [620-06];

q) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 108, 109, and 110, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively, [726];

r) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 108, 109, and 111, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively, [726-M101L];

s) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 41, 42, and 43, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 44, AAS, and 45, respectively,[140];

t) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 93, 94, and 95, respectively, and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 128, XAS, wherein X is D or G,and 129, respectively, [613/613-08];

u) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 46, 119, and 120, respectively; and a VL region comprising CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 49, AAS, and 50, respectively,[148/140];

v) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 123, 124, and 125, respectively; and a VL region comprising CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 60, GAS, and 61, respectively[171/172/181]; and

w) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 121, 109, and 122, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively [726/187]; and

x) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.:93, 126, and 127, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 96, GAS, and 97,respectively [613/608-01/610-01/620-06].

In one embodiment, the at least one binding region comprises a VH regionand a variable light chain (VL) region having, at most 5 mutations orsubstitutions selected from conservative, physical or functional aminoacids, such as at most 4 mutations or substitutions selected fromconservative, physical or functional amino acids, such as at most 3mutations or substitutions selected from conservative, physical orfunctional amino acids, such as at most 2 mutations selected fromconservative, physical or functional amino acids or substitutions, suchas at most 1 mutation or substitution selected from a conservative,physical or functional amino acid, in total across the CDR sequences insaid variable heavy chain and variable light chain selected from thegroup consisting of;

a) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 36, 37, and 38, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 39, GAS, and 40, respectively,[107];

b) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 46, 47, and 48, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 49, AAS, and 50, respectively,[148];

c) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 114, 115, and 116, respectively, and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 117, DAS, and 118,respectively [733];

d) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 51, 52, and 53, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 55, GAS, and 56, respectively[154];

e) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 51, 52, and 54, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 55, GAS, and 56, respectively[154-M1031];

f) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 57, 58, and 59, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 60, GAS, and 61, respectively,[171];

g) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 62, 63, and 64, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 65, GAS, and 66, respectively,[172];

h) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 67, 68, and 69, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 70, GAS, and 71, respectively,[181];

i) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 72, 73, and 75, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 76, ATS, and 77, respectively,[183];

j) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 72, 74, and 75, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 76, ATS, and 77, respectively,[183-N52Q];

k) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 78, 79, and 80, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 81, AAS, and 82, respectively,[187];

l) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 83, 84, and 85, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 86, GAS, and 87, respectively,[608-01];

m) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 88, 89, and 90, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 91, GAS, and 92, respectively,[610-01];

n) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 93, 94, and 95, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 96, GAS, and 97, respectively,[613];

o) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 98, 99, and 100, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 10, DAS, and 102,respectively, [613-08];

p) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 103, 104, and 105, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 106, GAS, and 107,respectively, [620-06];

q) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 108, 109, and 110, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively, [726];

r) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 108, 109, and 111, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively, [726-M101L];

s) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 41, 42, and 43, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 44, AAS, and 45, respectively,[140];

t) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 93, 94, and 95, respectively, and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 128, XAS, wherein X is D or G,and 129, respectively, [613/613-08];

u) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 46, 119, and 120, respectively; and a VL region comprising CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 49, AAS, and 50, respectively,[148/140];

v) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 123, 124, and 125, respectively; and a VL region comprising CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 60, GAS, and 61, respectively[171/172/181]; and

w) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 121, 109, and 122, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively [726/187]; and

x) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.:93, 126, and 127, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 96, GAS, and 97,respectively [613/608-01/610-01/620-06].

Hereby embodiments are provided wherein mutations or substitutions of upto five mutations or substitutions are allowed across the three CDRsequences in the variable heavy chain and variable light chain. The upto five mutations or substitutions may be distributed across the threeCDR sequences of the variable heavy chain and the three CDR sequences ofthe variable light chain. The up to five mutations or substitutions maybe distributed across the six CDR sequences of the binding region. Themutations or substitutions may be of conservative, physical orfunctional amino acids such that mutations or substitutions do notchange the epitope or preferably do not modify binding affinity to theepitope more than 30%, such as more than 20% or such as more than 10%.The conservative, physical or functional amino acids are selected fromthe 20 natural amino acids found i.e, Arg, His, Lys, Asp, Glu, Ser, Thr,Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr and Val.

In a particular embodiment, the at least one binding region comprises aVH region and a variable light chain (VL) region selected from the groupconsisting of;

a) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 36, 37, and 38, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 39, GAS, and 40, respectively,[107];

b) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 46, 47, and 48, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 49, AAS, and 50, respectively,[148];

c) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 114, 115, and 116, respectively, and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 117, DAS, and 118,respectively [733];

d) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 51, 52, and 53, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 55, GAS, and 56, respectively[154];

e) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 51, 52, and 54, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 55, GAS, and 56, respectively[154-M103L];

f) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 57, 58, and 59, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 60, GAS, and 61, respectively,[171];

g) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 62, 63, and 64, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 65, GAS, and 66, respectively,[172];

h) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 67, 68, and 69, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 70, GAS, and 71, respectively,[181];

i) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 72, 73, and 75, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 76, ATS, and 77, respectively,[183];

j) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 72, 74, and 75, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 76, ATS, and 77, respectively,[183-N52Q];

k) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 78, 79, and 80, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 81, AAS, and 82, respectively,[187];

l) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 83, 84, and 85, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 86, GAS, and 87, respectively,[608-01];

m) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 88, 89, and 90, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 91, GAS, and 92, respectively,[610-01];

n) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 93, 94, and 95, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 96, GAS, and 97, respectively,[613];

o) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 98, 99, and 100, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 10, DAS, and 102,respectively, [613-08];

p) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 103, 104, and 105, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 106, GAS, and 107,respectively, [620-06];

q) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 108, 109, and 110, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively, [726];

r) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 108, 109, and 111, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively, [726-M101L];

s) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 41, 42, and 43, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 44, AAS, and 45, respectively,[140];

t) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 93, 94, and 95, respectively, and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 128, XAS, wherein X is D or G,and 129, respectively, [613/613-08];

u) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 46, 119, and 120, respectively; and a VL region comprising CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 49, AAS, and 50, respectively,[148/140];

v) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 123, 124, and 125, respectively; and a VL region comprising CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 60, GAS, and 61, respectively[171/172/181]; and

w) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 121, 109, and 122, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively [726/187]; and

x) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.:93, 126, and 127, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 96, GAS, and 97,respectively [613/608-01/610-01/620-06].

In one embodiment, the at least one binding region comprises a VH regionand a VL region selected from the group consisting of;

a) a VH region comprising SEQ ID No: 1 and a VL region comprising SEQ IDNo: 2 [107];

b) a VH region comprising SEQ ID No: 5 and a VL region comprising SEQ IDNo: 6 [148];

c) a VH region comprising SEQ ID No: 34 and a VL region comprising SEQID No: 35 [733]

d) a VH region comprising SEQ ID No: 7 and a VL region comprising SEQ IDNo: 9 [154];

e) a VH region comprising SEQ ID No: 10 and a VL region comprising SEQID No: 11 [171];

f) a VH region comprising SEQ ID No: 16 and a VL region comprising SEQID No: 18 [183];

g) a VH region comprising SEQ ID No: 25 and a VL region comprising SEQID No: 26 [613];

h) a VH region comprising SEQ ID No: 31 and a VL region comprising SEQID No: 33 [726];

i) a VH region comprising SEQ ID No: 3 and a VL region comprising SEQ IDNo: 4 [140];

j) a VH region comprising SEQ ID No:8 and a VL region comprising SEQ IDNo:9 [154-M103L];

k) a VH region comprising SEQ ID No:12 and a VL region comprising SEQ IDNo:13 [172];

l) a VH region comprising SEQ ID No:14 and a VL region comprising SEQ IDNo:15 [181];

m) a VH region comprising SEQ ID No:17 and a VL region comprising SEQ IDNo:18 [183-N52Q];

n) a VH region comprising SEQ ID No:19 and a VL region comprising SEQ IDNo:20 [187];

o) a VH region comprising SEQ ID No:21 and a VL region comprising SEQ IDNo:22 [608-01];

p) a VH region comprising SEQ ID No:23 and a VL region comprising SEQ IDNo:24 [610-01];

q) a VH region comprising SEQ ID No:27 and a VL region comprising SEQ IDNo:28 [613-08];

r) a VH region comprising SEQ ID No:29 and a VL region comprising SEQ IDNo:30 [620-06]; and

s) a VH region comprising SEQ ID No:32 and a VL region comprising SEQ IDNo:33 [726-M101L].

In one embodiment, the at least one binding region comprises a variableheavy chain (VH) region and a variable light chain (VL) region having atmost 10 mutations or substitutions, at most 5 mutations orsubstitutions, such as at most 4 mutations or substitutions, such as atmost 3 mutations or substitutions, such as at most 2 mutations orsubstitutions, such as at most 1 mutation or substitution, across saidvariable heavy chain and variable light chain sequences selected fromthe group consisting of;

In one embodiment, the at least one binding region comprises a VH regionand a VL region selected from the group consisting of;

a) a VH region comprising SEQ ID No: 1 and a VL region comprising SEQ IDNo: 2 [107];

b) a VH region comprising SEQ ID No: 5 and a VL region comprising SEQ IDNo: 6 [148];

c) a VH region comprising SEQ ID No: 34 and a VL region comprising SEQID No: 35 [733]

d) a VH region comprising SEQ ID No: 7 and a VL region comprising SEQ IDNo: 9 [154];

e) a VH region comprising SEQ ID No: 10 and a VL region comprising SEQID No: 11 [171];

f) a VH region comprising SEQ ID No: 16 and a VL region comprising SEQID No: 18 [183];

g) a VH region comprising SEQ ID No: 25 and a VL region comprising SEQID No: 26 [613];

h) a VH region comprising SEQ ID No: 31 and a VL region comprising SEQID No: 33 [726];

i) a VH region comprising SEQ ID No: 3 and a VL region comprising SEQ IDNo: 4 [140];

j) a VH region comprising SEQ ID No:8 and a VL region comprising SEQ IDNo:9 [154-M1031];

k) a VH region comprising SEQ ID No:12 and a VL region comprising SEQ IDNo:13 [172];

l) a VH region comprising SEQ ID No:14 and a VL region comprising SEQ IDNo:15 [181];

m) a VH region comprising SEQ ID No:17 and a VL region comprising SEQ IDNo:18 [183-N52Q];

n) a VH region comprising SEQ ID No:19 and a VL region comprising SEQ IDNo:20 [187];

o) a VH region comprising SEQ ID No:21 and a VL region comprising SEQ IDNo:22 [608-01];

p) a VH region comprising SEQ ID No:23 and a VL region comprising SEQ IDNo:24 [610-01];

q) a VH region comprising SEQ ID No:27 and a VL region comprising SEQ IDNo:28 [613-08];

r) a VH region comprising SEQ ID No:29 and a VL region comprising SEQ IDNo:30 [620-06]; and

s) a VH region comprising SEQ ID No:32 and a VL region comprising SEQ IDNo:33 [726-M101L].

Hereby embodiments are provided wherein mutations or substitutions of upto 10 mutations or substitutions are allowed across the variable heavychain and variable light chain. The up to 10 mutations or substitutionsmay be distributed across the full length of the variable heavy chainand the variable light chain of each binding region. The mutations orsubstitutions may be of conservative, physical or functional amino acidssuch that the mutations or substitutions do not change the epitope andpreferably do not modify binding affinity to the epitope more than 30%,such as more than 20% or such as more than 10%. The conservative,physical or functional amino acids are selected from the 20 naturalamino acids found i.e. Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys,Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr and Val.

In one embodiment, the at least one binding region comprises a variableheavy chain (VH) region and a variable light chain (VL) region having atmost 10 mutations or substitutions selected from conservative, physicalor functional amino acids, at most 5 mutations or substitutions selectedfrom conservative, physical or functional amino acids, such as at most 4mutations or substitutions selected from conservative, physical orfunctional amino acids, such as at most 3 mutations or substitutionsselected from conservative, physical or functional amino acids, such asat most 2 mutations selected from conservative, physical or functionalamino acids or substitutions, such as at most 1 mutation or substitutionselected from a conservative, physical or functional amino acid, acrosssaid variable heavy chain and variable light chain sequences selectedfrom the group consisting of;

In one embodiment, the at least one binding region comprises a VH regionand a VL region selected from the group consisting of;

a) a VH region comprising SEQ ID No: 1 and a VL region comprising SEQ IDNo: 2 [107];

b) a VH region comprising SEQ ID No: 5 and a VL region comprising SEQ IDNo: 6 [148];

c) a VH region comprising SEQ ID No: 34 and a VL region comprising SEQID No: 35 [733]

d) a VH region comprising SEQ ID No: 7 and a VL region comprising SEQ IDNo: 9 [154];

e) a VH region comprising SEQ ID No: 10 and a VL region comprising SEQID No: 11 [171];

f) a VH region comprising SEQ ID No: 16 and a VL region comprising SEQID No: 18 [183];

g) a VH region comprising SEQ ID No: 25 and a VL region comprising SEQID No: 26 [613];

h) a VH region comprising SEQ ID No: 31 and a VL region comprising SEQID No: 33 [726];

i) a VH region comprising SEQ ID No: 3 and a VL region comprising SEQ IDNo: 4 [140];

j) a VH region comprising SEQ ID No:8 and a VL region comprising SEQ IDNo:9 [154-M103L];

k) a VH region comprising SEQ ID No:12 and a VL region comprising SEQ IDNo:13 [172];

l) a VH region comprising SEQ ID No:14 and a VL region comprising SEQ IDNo:15 [181];

m) a VH region comprising SEQ ID No:17 and a VL region comprising SEQ IDNo:18 [183-N52Q];

n) a VH region comprising SEQ ID No:19 and a VL region comprising SEQ IDNo:20 [187];

o) a VH region comprising SEQ ID No:21 and a VL region comprising SEQ IDNo:22 [608-01];

p) a VH region comprising SEQ ID No:23 and a VL region comprising SEQ IDNo:24 [610-01];

q) a VH region comprising SEQ ID No:27 and a VL region comprising SEQ IDNo:28 [613-08];

r) a VH region comprising SEQ ID No:29 and a VL region comprising SEQ IDNo:30 [620-06]; and

s) a VH region comprising SEQ ID No:32 and a VL region comprising SEQ IDNo:33 [726-M101L].

Hereby embodiments are provided wherein mutations or substitutions of upto 10 mutations or substitutions are allowed across the variable heavychain and variable light chain. The up to 10 mutations or substitutionsmay be distributed across the variable heavy chain and the variablelight chain. The up to 10 mutations or substitutions may be distributedacross the binding region. The mutations or substitutions may be ofconservative, physical or functional amino acids such that mutations orsubstitutions do not change the epitope or modify binding to theepitope.

In one aspect, the present invention relates to antibodies witch bindsto an extracellular domain of AXL without competing or interfering withGas6 binding to AXL. In a particular embodiment, the antibody binds tothe extracellular domain Ig1-like domain without competing orinterfering with Gas6 binding to AXL. In one embodiment, the antibodybinds to the extracellular domain Ig1-like and show no more than a 20%reduction in maximal Gas6 binding to AXL. In one embodiment, theantibody show no more than a 15% reduction in maximal Gas6 binding toAXL. In one embodiment, the antibody show no more than a 10% reductionin maximal Gas6 binding to AXL. In one embodiment, the antibody show nomore than a 5% reduction in maximal Gas6 binding to AXL. In oneembodiment, the antibody show no more than a 4% reduction in maximalGas6 binding to AXL In one embodiment, the antibody show no more than a2% reduction in maximal Gas6 binding to AXL. In one embodiment, theantibody show no more than a 1% reduction in maximal Gas6 binding. Inone embodiment the antibody binds to the extracellular domain Ig2-likedomain without competing or interfering with Gas6 binding to AXL. In oneembodiment, the antibody binds to the extracellular domain Ig2-like andshow no more than a 20%, such as no more than 15%, such as no more than10%, such as no more than 5%, such as no more than 4%, such as no morethan 2%, such as no more than 1%, reduction in maximal Gas6 binding toAXL. The embodiment's ability to compete with or reduce Gas6 binding maybe determined as disclosed in Example 2 or Example 12. In one embodimentthe antibody binds to the extracellular domain Ig2-like domain withoutcompeting or interfering with maximal Gas6 binding to AXL.

In one embodiment, the antibody binds to an epitope on AXL which epitopeis recognized by an antibody herein described.

Methods of determining an epitope to which an antibody binds iswell-known in the art, and thus, the skilled person would know how todetermine such an epitope. However, an example of determining whether anantibody binds within any epitope herein defined would be by pointmutations of the AXL extracellular domain. It is within the knowledge ofthe skilled person to introduce point mutation(s) in the AXLextracellular domain and test for antibody binding to point mutated AXLextracellular domains. When referring to amino acid positions within theAXL protein in the context of epitopes, the numbering has beendetermined as described in Example 7. Thus, numbering of amino acidpositions defining the epitope was done based on the sequences put forthin FIG. 6, i.e. the first amino acid in the shown sequence was numberedas position ‘1’, the second as position ‘2’, etc.

In one embodiment, the antibody binds to an epitope within the Ig1-likedomain of AXL, the epitope comprises or requires the amino acidscorresponding to positions L121 to Q129 or T112 to Q124 of human AXL. Inone embodiment, the antibody binds to an epitope within the Ig1-likedomain of AXL, the epitope comprises or requires one or more amino acidscorresponding to positions L121 to Q129 or T112 to Q124 of human AXL. Inone embodiment the epitope comprises one or more amino acid in positionL121, G122, H123, Q124, T125, F126, V127, 5128, Q129 or more amino acidin position T112, G113, Q114, Y115, Q116, C117, L118, V119, F120, L121,G122, H123, Q124.

In another embodiment, the antibody binds to an epitope within theIg2-like domain of AXL which epitope comprises or requires the aminoacids corresponding to position D170 or the combination of D179 and T182to R190 of human AXL.

In one embodiment, the antibody binds to an epitope within the Ig2-likedomain of AXL which epitope comprises or requires the amino acidscorresponding to position D170 or the combination of D179 and one ormore amino acids corresponding to positions T182 to R190 of human AXL.In one embodiment the epitope comprises one or more amino acid inposition T182, A183, P183, G184, H185, G186, P187, Q189, R190.

In another embodiment, the antibody binds to an epitope within theFN1-like domain of human AXL which epitope comprises or requires theamino acids corresponding to positions Q272 to A287 and G297 to P301 ofhuman AXL.

In another embodiment, the antibody binds to an epitope within theFN2-like domain of human AXL which epitope comprises or requires theamino acids corresponding to positions A359, R386, and Q436 to K439 ofhuman AXL.

In one embodiment, the antibody binds to an epitope within the FN1-likedomain of human AXL.

In one embodiment, the antibody binds to an epitope on AXL which epitopeis recognized by any one of the antibodies defined by

a)) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 36, 37, and 38, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 39, GAS, and 40, respectively,[107];

b) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 46, 47, and 48, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 49, AAS, and 50, respectively,[148];

c) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 114, 115, and 116, respectively, and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 117, DAS, and 118,respectively [733];

d) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 51, 52, and 53, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 55, GAS, and 56, respectively[154];

e) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 51, 52, and 54, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 55, GAS, and 56, respectively[154-M103L];

f) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 57, 58, and 59, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 60, GAS, and 61, respectively,[171];

g) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 62, 63, and 64, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 65, GAS, and 66, respectively,[172];

h) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 67, 68, and 69, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 70, GAS, and 71, respectively,[181];

i) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 72, 73, and 75, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 76, ATS, and 77, respectively,[183];

j) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 72, 74, and 75, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 76, ATS, and 77, respectively,[183-N52Q];

k) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 78, 79, and 80, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 81, AAS, and 82, respectively,[187];

l) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 83, 84, and 85, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 86, GAS, and 87, respectively,[608-01];

m) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 88, 89, and 90, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 91, GAS, and 92, respectively,[610-01];

n) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 93, 94, and 95, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 96, GAS, and 97, respectively,[613];

o) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 98, 99, and 100, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 10, DAS, and 102,respectively, [613-08];

p) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 103, 104, and 105, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 106, GAS, and 107,respectively, [620-06];

q) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 108, 109, and 110, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively, [726];

r) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 108, 109, and 111, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively, [726-M101L];

s) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 41, 42, and 43, respectively; and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 44, AAS, and 45, respectively,[140];

t) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 93, 94, and 95, respectively, and a VL region comprising the CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 128, XAS, wherein X is D or G,and 129, respectively, [613/613-08];

u) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 46, 119, and 120, respectively; and a VL region comprising CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 49, AAS, and 50, respectively,[148/140];

v) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 123, 124, and 125, respectively; and a VL region comprising CDR1,CDR2, and CDR3 sequences of SEQ ID Nos.: 60, GAS, and 61, respectively[171/172/181]; and

w) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.: 121, 109, and 122, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 112, AAS, and 113,respectively [726/187]; and

x) a VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNos.:93, 126, and 127, respectively; and a VL region comprising theCDR1, CDR2, and CDR3 sequences of SEQ ID Nos.: 96, GAS, and 97,respectively [613/608-01/610-01/620-06].

In one embodiment, the antibody comprises a heavy chain of an isotypeselected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

The term “isotype” as used herein refers to the immunoglobulin class(for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or anyallotypes thereof, such as IgG1m(za) and IgG1m(0) that is encoded byheavy chain constant region genes. Further, each heavy chain isotype canbe combined with either a kappa (κ) or lambda (λ) light chain.

In one embodiment, the isotype is IgG1, optionally allotype IgG1m(f).

In one embodiment, the antibody is a full-length monoclonal antibody,optionally a full-length monoclonal IgG1,κ antibody.

The term “full-length antibody” when used herein, refers to an antibody(e.g., a parent or variant antibody) which contains all heavy and lightchain constant and variable domains corresponding to those that arenormally found in a wild-type antibody of that isotype. A full-lengthantibody according to the present invention may be produced by a methodcomprising the steps of (i) cloning the CDR sequences into a suitablevector comprising complete heavy chain sequences and complete lightchain sequence, and (ii) expressing the complete heavy and light chainsequences in suitable expression systems. It is within the knowledge ofthe skilled person to produce a full-length antibody when starting outfrom either CDR sequences or full variable region sequences. Thus, theskilled person would know how to generate a full-length antibodyaccording to the present invention.

In one embodiment, the antibody is a human antibody.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and framework regions derived from humangermline immunoglobulin sequences and a human immunoglobulin constantdomain. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations, insertions or deletions introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody”, as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another non-humanspecies, such as a mouse, have been grafted onto human frameworksequences.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, for instance by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library, and wherein the selected human antibody isat least 90%, such as at least 95%, for instance at least 96%, such asat least 97%, for instance at least 98%, or such as at least 99%identical in amino acid sequence to the amino acid sequence encoded bythe germline immunoglobulin gene. Typically, outside the heavy chainCDR3, a human antibody derived from a particular human germline sequencewill display no more than 20 amino acid differences, e.g. no more than10 amino acid differences, such as no more than 9, 8, 7, 6 or 5, forinstance no more than 4, 3, 2, or 1 amino acid difference from the aminoacid sequence encoded by the germline immunoglobulin gene.

The antibody according to the present invention may comprise amino acidmodifications in the immunoglobulin heavy and/or light chains. In aparticular embodiment, amino acids in the Fc region of the antibody maybe modified.

The term “Fc region” as used herein, refers to a region comprising, inthe direction from the N- to C-terminal end of the antibody, at least ahinge region, a CH2 region and a CH3 region. An Fc region of theantibody may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (such aseffector cells) and components of the complement system.

The term “hinge region” as used herein refers to the hinge region of animmunoglobulin heavy chain. Thus, for example the hinge region of ahuman IgG1 antibody corresponds to amino acids 216-230 according to theEu numbering as set forth in Kabat [26]. However, the hinge region mayalso be any of the other subtypes as described herein.

The term “CH1 region” or “CH1 domain” as used herein refers to the CH1region of an immunoglobulin heavy chain. Thus, for example the CH1region of a human IgG1 antibody corresponds to amino acids 118-215according to the Eu numbering as set forth in Kabat [26]. However, theCH1 region may also be any of the other subtypes as described herein.

The term “CH2 region” or “CH2 domain” as used herein refers to the CH2region of an immunoglobulin heavy chain. Thus, for example the CH2region of a human IgG1 antibody corresponds to amino acids 231-340according to the Eu numbering as set forth in Kabat [26]. However, theCH2 region may also be any of the other subtypes as described herein.

The term “CH3 region” or “CH3 domain” as used herein refers to the CH3region of an immunoglobulin heavy chain. Thus for example the CH3 regionof a human IgG1 antibody corresponds to amino acids 341-447 according tothe Eu numbering as set forth in Kabat [26]. However, the CH3 region mayalso be any of the other subtypes as described herein.

In another embodiment, the antibody is an effector-function-deficientantibody, a stabilized IgG4 antibody or a monovalent antibody.

In one particular embodiment, the heavy chain has been modified suchthat the entire hinge region has been deleted.

In one embodiment, the sequence of the antibody has been modified sothat it does not comprise any acceptor sites for N-linked glycosylation.

In one embodiment, the antibody is a single-chain antibody.

In further aspect, the present invention relates to a multispecificantibody comprising at least a first binding region of an antibodyaccording to any aspect or embodiment herein described, and a secondbinding region which binds a different target or epitope than the firstbinding region. The term “multispecific antibody” as used herein, refersto antibodies wherein the binding regions two to at least two, such asat least three, different antigens or at least two, such as at leastthree, different epitopes on the same antigen.

In one embodiment, the present invention relates to a bispecificantibody comprising a first binding region of an antibody according toany aspect or embodiments herein described, and a second binding regionwhich binds a different target or epitope than the first binding region.

The term “bispecific” as used herein, refers to binding molecules, suchas antibodies wherein the binding regions of the binding molecule bindto two different antigens or two different epitopes on the same antigen.

The term “bispecific antibody” refers to an antibody havingspecificities for at least two different, typically non-overlapping,epitopes. Such epitopes may be on the same or different targets. If theepitopes are on different targets, such targets may be on the same cellor different cells, cell types or structures, such as extracellulartissue.

The term “different target” as used herein, refers to another protein,molecule or the like than AXL or an AXL fragment.

Examples of bispecific antibody molecules which may be used in thepresent invention comprise (i) a single antibody that has two armscomprising different antigen-binding regions, (ii) a single chainantibody that has specificity to two different epitopes, e.g., via twoscFvs linked in tandem by an extra peptide linker; (iii) adual-variable-domain antibody (DVD-Ig™), where each light chain andheavy chain contains two variable domains in tandem through a shortpeptide linkage [29]; (iv) a chemically-linked bispecific (Fab′)2fragment; (v) a Tandab®, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (vi) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(vii) a so called “dock and lock” molecule (Dock-and-Lock®), based onthe “dimerization and docking domain” in Protein Kinase A, which, whenapplied to Fabs, can yield a trivalent bispecific binding proteinconsisting of two identical Fab fragments linked to a different Fabfragment; (viii) a so-called Scorpion molecule, comprising, e.g., twoscFvs fused to both termini of a human Fab-arm; and (ix) a diabody.

In one embodiment, the bispecific antibody of the present invention is adiabody, a cross-body, such as CrossMabs, or a bispecific antibodyobtained via a controlled Fab arm exchange (such as described in [30]).

Examples of different classes of bispecific antibodies include but arenot limited to (i) IgG-like molecules with complementary CH3 domains toforce heterodimerization; (ii) recombinant IgG-like dual targetingmolecules, wherein the two sides of the molecule each contain the Fabfragment or part of the Fab fragment of at least two differentantibodies; (iii) IgG fusion molecules, wherein full length IgGantibodies are fused to extra Fab fragment or parts of Fab fragment;(iv) Fc fusion molecules, wherein single chain Fv molecules orstabilized diabodies are fused to heavy-chain constant-domains,Fc-regions or parts thereof; (v) Fab fusion molecules, wherein differentFab-fragments are fused together, fused to heavy-chain constant-domains,Fc-regions or parts thereof; and (vi) ScFv- and diabody-based and heavychain antibodies (e.g., domain antibodies, Nanobodies®) whereindifferent single chain Fv molecules or different diabodies or differentheavy-chain antibodies (e.g. domain antibodies, Nanobodies®) are fusedto each other or to another protein or carrier molecule fused toheavy-chain constant-domains, Fc-regions or parts thereof.

Examples of IgG-like molecules with complementary CH3 domains moleculesinclude but are not limited to the Triomab® (Trion Pharma/FreseniusBiotech, [31]), the Knobs-into-Holes (Genentech, [32]), CrossMAbs(Roche, [33]) and the electrostatically-matched (Amgen, [34] and [35];Chugai, [36]; Oncomed, [37]), the LUZ-Y (Genentech), DIG-body andPIG-body (Pharmabcine), the Strand Exchange Engineered Domain body(SEEDbody)(EMD Serono, [38]), the Biclonics (Merus), FcΔAdp (Regeneron,[39]), bispecific IgG1 and IgG2 (Pfizer/Rinat, [40]), Azymetric scaffold(Zymeworks/Merck, [41]), mAb-Fv (Xencor, [42]), bivalent bispecificantibodies (Roche [43]) and DuoBody® molecules (Genmab A/S, [30]).

Examples of recombinant IgG-like dual targeting molecules include butare not limited to Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-oneAntibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2(F-Star, [44]), Zybodies™ (Zyngenia), approaches with common light chain(Crucell/Merus, [45]), κλBodies (Novlmmune) and CovX-body (CovX/Pfizer).

Examples of IgG fusion molecules include but are not limited to DualVariable Domain (DVD)-Ig™ (Abbott, [46]), Dual domain double headantibodies (Unilever; Sanofi Aventis, [47]), IgG-like Bispecific(ImClone/Eli Lilly), Ts2Ab (Medlmmune/AZ) and BsAb (Zymogenetics),HERCULES (Biogen Idec, [48]), scFv fusion (Novartis), scFv fusion(Changzhou Adam Biotech Inc, [49]) and TvAb (Roche, [50], [51]).

Examples of Fc fusion molecules include but are not limited to ScFv/FcFusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion,Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART™)(MacroGenics, [52], [53]) and Dual(ScFv)2-Fab (National Research Centerfor Antibody Medicine—China).

Examples of Fab fusion bispecific antibodies include but are not limitedto F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech),Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) andFab-Fv (UCB-Celltech).

Examples of ScFv-, diabody-based and domain antibodies include but arenot limited to Bispecific T Cell Engager (BiTE®) (Micromet, TandemDiabody (Tandab™) (Affimed), Dual Affinity Retargeting Technology (DART)(MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies(AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) andCOMBODY (Epigen Biotech), dual targeting Nanobodies® (Ablynx), dualtargeting heavy chain only domain antibodies.

A bispecific antibody according the present invention may be generatedby introducing modifications in the constant region of the antibody.

Unless otherwise stated or contradicted by context, the amino acids ofthe constant region sequences are herein numbered according to theEu-index of numbering (described in [26]). The terms “Eu-index ofnumbering” and “Eu numbering as set forth in Kabat” may be usedinterchangeably and have the same meaning and purpose. Thus, an aminoacid or segment in one sequence that “corresponds to” an amino acid orsegment in another sequence is one that aligns with the other amino acidor segment using a standard sequence alignment program such as ALIGN,ClustalW or similar, typically at default settings and has at least 50%,at least 80%, at least 90%, or at least 95% identity to a human IgG1heavy chain. It is well-known in the art how to align a sequence orsegment in a sequence and thereby determine the corresponding positionin a sequence to an amino acid position according to the presentinvention.

The term “amino acid corresponding to positions” as used herein refersto an amino acid position number in a human IgG1 heavy chain.

The present invention also provides antibodies comprising functionalvariants of the VL region, VH region, or one or more CDRs of theantibodies of the examples. A functional variant of a VL, VH, or CDRused in the context of an AXL antibody still allows the antibody toretain at least a substantial proportion (at least about 50%, 60%, 70%,80%, 90%, 95%, 99% or more) of the affinity/avidity and/or thespecificity/selectivity of the parent antibody and in some cases such anAXL antibody may be associated with greater affinity, selectivity and/orspecificity than the parent antibody.

Such functional variants typically retain significant sequence identityto the parent antibody. The percent identity between two sequences is afunction of the number of identical positions shared by the sequences(i.e., % homology=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences may be accomplished using a mathematicalalgorithm, which is well-known in the art.

The VH, VL and/or CDR sequences of variants may differ from those of theparent antibody sequences through mostly conservative substitutions; forinstance at least about 35%, about 50% or more, about 60% or more, about70% or more, about 75% or more, about 80% or more, about 85% or more,about 90% or more, (e.g., about 65-95%, such as about 92%, 93% or 94%)of the substitutions in the variant are conservative amino acid residuereplacements.

The VH, VL and/or CDR sequences of variants may differ from those of theparent antibody sequences through mostly conservative substitutions; forinstance 10 or less, such as 9 or less, 8 or less, 7 or less, 6 or less,5 or less, 4 or less, 3 or less, 2 or less or 1 of the substitutions inthe variant are conservative amino acid residue replacements.

The term “amino acid” and “amino acid residue” may herein be usedinterchangeably, and are not to be understood limiting.

In the context of the present invention, the amino acid may be definedby conservative or non-conservative amino acids, and may therefore beclassified accordingly. Amino acid residues may also be divided intoclasses defined by alternative physical and functional properties. Thus,classes of amino acids may be reflected in one or both of the followingtables:

Amino acid residue of conservative class Acidic Residues D and E BasicResidues K, R, and H Hydrophilic Uncharged Residues S, T, N, and QAliphatic Uncharged Residues G, A, V, L, and I Non-polar UnchargedResidues C, M, and P Aromatic Residues F, Y, and W

Alternative Physical and Functional Classifications of Amino AcidResidues Alcohol group-containing residues S and T Aliphatic residues I,L, V, and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobicresidues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively chargedresidues D and E Polar residues C, D, E, H, K, N, Q, R, S, and TPositively charged residues H, K, and R Small residues A, C, D, G, N, P,S, T, and V Very small residues A, G, and S Residues involved in turnformation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q,T, K, S, G, P, D, E, and R

In the context of the present invention, a substitution in an antibodyis indicated as:

Original amino acid—position—substituted amino acid;

Referring to the well-recognized nomenclature for amino acids, the threeletter code, or one letter code, is used, including the codes “Xaa” or“X” to indicate any amino acid residue. Thus, Xaa or X may typicallyrepresent any of the 20 naturally occurring amino acids. The term“naturally occurring” as used herein refers to any one of the followingamino acid residues; glycine, alanine, valine, leucine, isoleucine,serine, threonine, lysine, arginine, histidine, aspartic acid,asparagine, glutamic acid, glutamine, proline, tryptophan,phenylalanine, tyrosine, methionine, and cysteine. Accordingly, thenotation “K409R” or “Lys409Arg” means, that the antibody comprises asubstitution of Lysine with Arginine in amino acid position 409.

Substitution of an amino acid at a given position to any other aminoacid is referred to as:

Original amino acid—position; or e.g. “K409”

For a modification where the original amino acid(s) and/or substitutedamino acid(s) may comprise more than one, but not all amino acid(s), themore than one amino acid may be separated by “,” or “/”. E.g. thesubstitution of Lysine with Arginine, Alanine, or Phenylalanine inposition 409 is:

“Lys409Arg,Ala,Phe” or “Lys409Arg/Ala/Phe” or “K409R,A,F” or “K409R/A/F”or “K409 to R, A, or F”.

Such designation may be used interchangeably in the context of theinvention but have the same meaning and purpose.

Furthermore, the term “a substitution” embraces a substitution into anyone or the other nineteen natural amino acids, or into other aminoacids, such as non-natural amino acids. For example, a substitution ofamino acid K in position 409 includes each of the followingsubstitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L,409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. This is,by the way, equivalent to the designation 409X, wherein the X designatesany amino acid other than the original amino acid. These substitutionsmay also be designated K409A, K409C, etc. or K409A,C, etc. orK409A/C/etc. The same applies by analogy to each and every positionmentioned herein, to specifically include herein any one of suchsubstitutions.

The antibody according to the invention may also comprise a deletion ofan amino acid residue. Such deletion may be denoted “del”, and includes,e.g., writing as K409del. Thus, in such embodiments, the Lysine inposition 409 has been deleted from the amino acid sequence.

In one particular embodiment, the bispecific antibody comprises a firstand a second heavy chain, each of the first and second heavy chaincomprises at least a hinge region, a CH2 and CH3 region, wherein in thefirst heavy chain at least one of the amino acids in the positionscorresponding to positions selected from the group consisting of K409,T366, L368, K370, D399, F405, and Y407 in a human IgG1 heavy chain hasbeen substituted, and in the second heavy chain at least one of theamino acids in the positions corresponding to a position selected fromthe group consisting of F405, T366, L368, K370, D399, Y407, and K409 ina human IgG1 heavy chain has been substituted, and wherein the first andthe second heavy chains are not substituted in the same positions.

In one embodiment, in the first heavy chain the amino acid in theposition corresponding to K409 in a human IgG1 heavy chain is not K, Lor M and optionally the amino acid in the position corresponding to F405in a human IgG1 heavy chain is F, and in the second heavy chain theamino acid in the position corresponding to F405 in a human IgG1 heavychain is not F and the amino acid in the position corresponding to K409in a human IgG1 heavy chain is K.

In one embodiment, in the first heavy chain, the amino acid in theposition corresponding to F405 in a human IgG1 heavy chain is not F, R,and G, and in the second heavy chain the amino acids in the positionscorresponding to a position selected from the group consisting of; T366,L368, K370, D399, Y407, and K409 in a human IgG1 heavy chain has beensubstituted.

In one embodiment, the amino acid in position corresponding to K409 in ahuman IgG1 heavy chain is another than K, L or M in the first heavychain, and in the second heavy chain the amino acid in positioncorresponding to F405 in a human IgG1 heavy chain is not F andoptionally the amino acid in the position corresponding to K409 in ahuman IgG1 heavy chain is K.

In one embodiment, the amino acid in the position corresponding to F405in a human IgG1 heavy chain is L in said first heavy chain, and theamino acid in the position corresponding to K409 in a human IgG1 heavychain is R in said second heavy chain, or vice versa.

Thus, in one embodiment, the amino acid in the position corresponding toK409 in a human IgG1 heavy chain is R in the first heavy chain, and theamino acid in the position corresponding to F405 in a human IgG1 heavychain is L in the second heavy chain.

In another embodiment, both the first and the second binding region ofthe bispecific antibody bind AXL. However, the first binding regioncomprises a different set of CDR sequences than the second bindingregion. Thus, in a particular embodiment, the bispecific antibodycomprising a first and a second binding region, and a first and a secondheavy chain, wherein the first and the second binding regions eachcomprise a VH and VL region selected from the group consisting of;

-   -   a) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 46, 47, and 48, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        49, AAS, and 50, respectively, [148];    -   b) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 114, 115, and 116, respectively, and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        117, DAS, and 118, respectively [733];    -   c) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 41, 42, and 43, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        44, AAS, and 45, respectively, [140];    -   d) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 51, 52, and 55, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        55, GAS, and 56, respectively. [154];    -   e) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 51, 52, and 54, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        55, GAS, and 56, respectively. [154-M103L];    -   f) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 57, 58, and 59, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        60, GAS, and 61, respectively, [171];    -   g) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 62, 63, and 64, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        65, GAS, and 66, respectively, [172];    -   h) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 67, 68, and 69, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        70, GAS, and 71, respectively, [181];    -   i) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 72, 73, and 75, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        76, ATS, and 77, respectively, [183];    -   j) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 72, 74, and 75, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        76, ATS, and 77, respectively, [183-N52Q];    -   k) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 78, 79, and 80, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        81, AAS, and 82, respectively, [187];    -   l) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 83, 84, and 85, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        86, GAS, and 87, respectively, [608-01];    -   m) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 88, 89, and 90, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        91, GAS, and 92, respectively, [610-01];    -   n) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 94, 95, and 95, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        96, GAS, and 97, respectively, [613];    -   o) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 98, 99, and 100, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        101, DAS, and 102, respectively, [613-08];    -   p) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 103, 104, and 105, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        106, GAS, and 107, respectively, [620-06];    -   q) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 108, 109, and 110, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        112, AAS, and 113, respectively, [726];    -   r) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 36, 37, and 38, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 39, GAS, and 40, respectively, [107]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 108, 109, and 111, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        112, AAS, and 113, respectively, [726-M101L];    -   s) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 114, 115, and 116, respectively, and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        117, DAS, and 118, respectively [733];    -   t) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 41, 42, and 43, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        44, AAS, and 45, respectively, [107];    -   u) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 51, 52, and 55, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        55, GAS, and 56, respectively. [154];    -   v) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 51, 52, and 54, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        55, GAS, and 56, respectively. [154-M103L];    -   w) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 57, 58, and 59, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        60, GAS, and 61, respectively, [171];    -   x) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 62, 63, and 64, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        65, GAS, and 66, respectively, [172];    -   y) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 67, 68, and 69, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        70, GAS, and 71, respectively, [181];    -   z) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 72, 73, and 75, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        76, ATS, and 77, respectively, [183];    -   aa) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 72, 74, and 75, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        76, ATS, and 77, respectively, [183-N52Q];    -   bb) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 78, 79, and 80, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        81, AAS, and 82, respectively, [187];    -   cc) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 83, 84, and 85, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        86, GAS, and 87, respectively, [608-01];    -   dd) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 88, 89, and 90, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        91, GAS, and 92, respectively, [610-01];    -   ee) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 94, 95, and 95, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        96, GAS, and 97, respectively, [613];    -   ff) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 98, 99, and 100, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        101, DAS, and 102, respectively, [613-08];    -   gg) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 103, 104, and 105, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        106, GAS, and 107, respectively, [620-06];    -   hh) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 108, 109, and 110, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        112, AAS, and 113, respectively, [726];    -   ii) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 46, 47, and 48, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 49, AAS, and 50, respectively, [148]; and a second        VH region comprising the CDR1, CDR2, and CDR3 sequences of SEQ        ID Nos.: 108, 109, and 111, respectively; and a second VL region        comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID Nos.:        112, AAS, and 113, respectively, [726-M101L];    -   jj) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 41, 42, and 43, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 44, AAS, and 45, respectively, [140];    -   kk) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 51, 52, and 55, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 55, GAS, and 56, respectively. [154];    -   ll) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 51, 52, and 54, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 55, GAS, and 56, respectively. [154-M103L];    -   mm) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 57, 58, and 59, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 60, GAS, and 61, respectively, [171];    -   nn) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 62, 63, and 64, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 65, GAS, and 66, respectively, [172];    -   oo) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 67, 68, and 69, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 70, GAS, and 71, respectively, [181];    -   pp) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 72, 73, and 75, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 76, ATS, and 77, respectively, [183];    -   qq) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 72, 74, and 75, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 76, ATS, and 77, respectively, [183-N52Q];    -   rr) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 78, 79, and 80, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 81, AAS, and 82, respectively, [187];    -   ss) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 83, 84, and 85, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 86, GAS, and 87, respectively, [608-01];    -   tt) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 88, 89, and 90, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 91, GAS, and 92, respectively, [610-01];    -   uu) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 94, 95, and 95, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 96, GAS, and 97, respectively, [613];    -   vv) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 98, 99, and 100, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 101, DAS, and 102, respectively, [613-08];    -   ww) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 103, 104, and 105, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 106, GAS, and 107, respectively, [620-06];    -   xx) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 108, 109, and 110, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 112, AAS, and 113, respectively, [726]; and    -   yy) a first VH region comprising the CDR1, CDR2, and CDR3        sequences of SEQ ID Nos.: 114, 115, and 116, respectively; and a        first VL region comprising the CDR1, CDR2, and CDR3 sequences of        SEQ ID Nos.: 117, DAS, and 118, respectively, [733]; and a        second VH region comprising the CDR1, CDR2, and CDR3 sequences        of SEQ ID Nos.: 108, 109, and 111, respectively; and a second VL        region comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID        Nos.: 112, AAS, and 113, respectively, [726-M101L];        Anti-AXL Antibody Drug Conjugate—Immunoconjugates

The antibodies according to any aspect or embodiment of the presentinvention may be conjugated to a therapeutic or diagnostic moiety, suchas a cytotoxic agent, a chemotherapeutic drug, a cytokine, animmunosuppressant, antibiotic, or a radioisotope. Such conjugates arereferred to herein as “immunoconjugates”. Immunoconjugates which includeone or more cytotoxins are referred to as “immunotoxins”. Antibodiesconjugated to a cytotoxic agent, drug or the like are also known asantibody-drug conjugates (ADC). An immunoconjugate may have a half-lifeof sufficient periods of time for the antibody-drug conjugate to beinternalized, degraded and induce cell killing by the released toxin.

Thus, in another aspect, the present invention relates to animmunoconjugate comprising the antibody according to any aspect orembodiment herein described, or a bispecific antibody according to anyaspect or embodiment herein described, and a therapeutic moiety, such asa cytotoxic agent, a chemotherapeutic drug, a cytokine, animmunosuppressant, antibiotic, or a radioisotope. The cytotoxic agent,chemotherapeutic drug, cytokine, immunosuppressant, antibiotic, orradioisotope may be conjugated to the antibody or the bispecificantibody via a linker.

ADCs are often designed such that the cytotoxic payload is inactive whenconjugated to the antibody. The cytotoxic payload may be releasedintracellularly upon internalization of the ADC after binding to theplasma-membrane of cells, or alternatively in response to proteolyticactivity in the tumor microenvironment. The term “internalized” or“internalization” as used herein, refers to a biological process inwhich molecules such as the antibody according to the present invention,are engulfed by the cell membrane and drawn into the interior of thecell. It may also be referred to as “endocytosis”.

Thus, the antibodies according to any aspect or embodiment of thepresent invention may be internalized into the cell upon binding to thetarget, AXL.

In some instances it may be desired to use antibodies which undergointernalization. Such antibodies that have good internalizationproperties may be suited for conjugation to a cytotoxic agent, drug, orthe like, optionally via a linker, which is designed to be cleavedintracellularly.

Once internalized, the ADC may be delivered to lysosomes in most cases,where effective drug release takes advantage of the catabolicenvironment found with these organelles. It is typically a linker thatconnects the antibody with a cytotoxic agent. Thus, specialized linkershave been designed to be cleaved only in a specific microenvironmentfound in or on the target tumor cell or in the tumor microenvironment.Examples include linkers that are cleaved by acidic conditions, reducingconditions, or specific proteases.

Stability of the antibody-linker-drug in circulation is importantbecause this allows antibody-mediated delivery of the drug to specifictarget cells. In addition, the long circulating half-life of the ADCprovides exposure for several days to weeks post injection. Drugs thatare conjugated through non-cleavable linkers and protease-cleavablelinkers are generally more stable in circulation than disulfide andhydrazone linkers, although the stability of the latter two linkers canbe tuned by altering the neighboring chemical structure [6].

In one embodiment, the therapeutic moiety is a cytotoxic agent.

A cytotoxin or cytotoxic agent includes any agent that is detrimental to(e.g., kills) cells. Suitable cytotoxic agents for formingimmunoconjugates of the present invention include taxol, tubulysins,duostatins, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, maytansine or ananalog or derivative thereof, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin; calicheamicin or analogs or derivativesthereof; antimetabolites (such as methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine,hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents(such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatinand other platinum derivatives, such as carboplatin; as well asduocarmycin A, duocarmycin SA, CC-1065 (a.k.a. rachelmycin), or analogsor derivatives of CC-1065), dolastatin, auristatin, pyrrolo[2,1-c][1,4]benzodiazepins (PDBs), indolinobenzodiazepine (IGNs) or analoguesthereof, antibiotics (such as dactinomycin (formerly actinomycin),bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin,mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)),anti-mitotic agents (e.g., tubulin-targeting agents), such as diphtheriatoxin and related molecules (such as diphtheria A chain and activefragments thereof and hybrid molecules); ricin toxin (such as ricin A ora deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin(SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussistoxin, tetanus toxin, soybean Bowman-Birk protease inhibitor,Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S),Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycintoxins. Other suitable conjugated molecules include antimicrobial/lyticpeptides such as CLIP, Magainin 2, mellitin, Cecropin, and P18;ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, diphtherin toxin, and Pseudomonas endotoxin. See, forexample, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. ACancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that maybe administered in combination with anti-AXL antibodies or antibody-drugconjugates of the present invention as described elsewhere herein, suchas, e.g., anti-cancer cytokines or chemokines, are also candidates fortherapeutic moieties useful for conjugation to an antibody disclosed inthe present invention.

The term “cytotoxic agent” as used herein, refers to any agent that isdetrimental to (e.g., kills) cells. For a description of these classesof drugs which are well known in the art, and their mechanisms ofaction, see [54]. Additional techniques relevant to the preparation ofantibody immunotoxins are provided in for instance [55] and [56].

In one embodiment, the cytotoxic agent is linked to said antibody, orfragment thereof, with a cleavable linker, such as N-succinimydyl4-(2-pyridyldithio)-pentanoate (SSP),maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PAB)or AV-1 K-lock valine-citrulline.

The term “cleavable linker” as used herein, refers to a subset oflinkers that are catalyzed by specific proteases in the targeted cell orin the tumor microenvironment, resulting in release of the cytotoxicagent. Examples of cleavable linkers are linkers based on chemicalmotifs including disulfides, hydrazones or peptides. Another subset ofcleavable linker, adds an extra linker motif between the cytotoxic agentand the primary linker, i.e. the site that attaches the linker-drugcombination to the antibody. In some embodiments, the extra linker motifis cleavable by a cleavable agent that is present in the intracellularenvironment (e. g. within a lysosome or endosome or caveola). The linkercan be, e. g. a peptidyl linker that is cleaved by an intracellularpeptidase or protease enzyme, including but not limited to, a lysosomalor endosomal protease. In some embodiments, the peptidyl linker is atleast two amino acids long or at least three amino acids long. Cleavingagents can include cathepsins B and D and plasmin, all of which areknown to hydrolyze dipeptide drug derivatives resulting in the releaseof active drug inside the target cells (see e. g. Dubowchik and Walker,1999, Pharm. Therapeutics 83:67-123). In a specific embodiment, thepeptidyl linker cleavable by an intracellular protease is a Val-Cit(valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker(see e.g. U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the Val-Cit linker). An advantage of usingintracellular proteolytic release of the therapeutic agent is that theagent is typically attenuated when conjugated and the serum stabilitiesof the conjugates are typically high.

In another embodiment, the cytotoxic agent is linked to said antibody,or fragment thereof, with a non-cleavable linker, such assuccinimidyl-4(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC) ormaleimidocaproyl (MC).

The term “noncleavable linker” as used herein, refers to a subset oflinkers which, in contrast to cleavable linkers, do not comprise motifsthat are specifically and predictably recognized by intracellular orextracellular proteases. Thus, ADCs based on non-cleavable linkers arenot released or cleaved form the antibody until the completeantibody-linker-drug complex is degraded in the lysosomal compartment.Examples of a non-cleavable linker are thioethers. In yet anotherembodiment, the linker unit is not cleavable and the drug is released byantibody degradation (see [57]). Typically, such a linker is notsubstantially sensitive to the extracellular environment. As usedherein, “not substantially sensitive to the extracellular environment”in the context of a linker means that no more than 20%, typically nomore than about 15%, more typically no more than about 10%, and evenmore typically no more than about 5%, no more than about 3%, or no morethan about 1% of the linkers, in a sample of antibody drug conjugatecompound, are cleaved when the antibody drug conjugate compound ispresent in an extracellular environment (e.g. plasma). Whether a linkeris not substantially sensitive to the extracellular environment can bedetermined for example by incubating with plasma the antibody drugconjugate compound for a predetermined time period (e.g. 2, 4, 8, 16 or24 hours) and then quantitating the amount of free drug present in theplasma.

In one embodiment, cytotoxic agent is selected from the group:DNA-targeting agents, e.g. DNA alkylators and cross-linkers, such ascalicheamicin, duocarmycin, rachelmycin (CC-1065), pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), and indolinobenzodiazepine (IGN);microtubule-targeting agents, such as duostatin, such as duostatin-3,auristatin, such as monomethylauristatin E (MMAE) andmonomethylauristatin F (MMAF), dolastatin, maytansine,N(2′)-deacetyl-N(2′)-(3-marcapto-1-oxopropyl)-maytansine (DM1), andtubulysin; and nucleoside analogs; or an analogs, derivatives, orprodrugs thereof.

In one embodiment, the immunoconjugate comprise a combination of;

i) the cytotoxic agent and said cleavable linker having bystander killcapacity;

ii) the cytotoxic agent and said cleavable linker not having bystanderkill capacity;

iii) the cytotoxic agent and said non-cleavable linker having bystanderkill capacity; or

iv) the cytotoxic agent and said non-cleavable linker not havingbystander kill capacity.

The term “bystander killing effect”, “bystander kill”, “bystander killcapacity” or “bystander cytotoxicity” as used herein, refers to theeffect where the cytotoxic agent that is conjugated to the antibody byeither a cleavable or non-cleavable linker has the capacity to diffuseacross cell membranes after the release from the antibody and therebycause killing of neighboring cells. When the cytotoxic agent isconjugated by a cleavable or non-cleavable linker, it may be either thecytotoxic agent only or the cytotoxic agent with a part of the linkerthat has the bystander kill capacity. The capacity to diffuse acrosscell membranes is related to the hydrophobicity of the the cytotoxicagent or the combination of the cytotoxic agent and the linker. Suchcytotoxic agents may advantageously be membrane-permeable toxins, suchas MMAE that has been released from the antibody by proteases.Especially in tumors with heterogeneous target expression and in solidtumors where antibody penetration may be limited, a bystander killingeffect may be desirable.

The term “no bystander kill capacity”, “no bystander killing effect”,“no-bystander kill” or “no bystander cytotoxicity” as used herein,refers to the effect where the cytotoxic agent that is conjugated to theantibody by either a cleavable or non-cleavable linker does not have thecapacity to diffuse across cell membranes after release from theantibody. Thus, such cytotoxic agents or combinations of the cytotoxicagent with the linker, will not be able to kill neighboring cells uponrelease from the antibody. It is believed without being bound by theory,that such combinations of a cytotoxic agent and either a cleavable ornon-cleavable linker will only kill cells expressing the target that theantibody binds.

A stable link between the antibody and cytotoxic agent is an importantfactor of an ADC. Both cleavable and non-cleavable types of linkers havebeen proven to be safe in preclinical and clinical trials.

In one embodiment, the cytotoxic agent is chosen from the group ofmicrotubule targeting agents, such as auristatins and maytansinoids.

The term “microtubule-targeting agent” as used herein, refers to anagent or drug which inhibits mitosis (cell division). Microtubules arestructures that are essential for proper separation of DNA during celldivision, and microtubule function critically depends on ‘dynamicinstability’, i.e. the process in which microtubule structures arecontinuously elongated and shortened. Microtubule-targeting agentsdisrupt or stabilize microtubules, which prevents formation of themitotic spindle, resulting in mitotic arrest and apoptosis. Themicrotubule-targeting agents can be derived from e.g. natural substancessuch as plant alkaloids, and prevent cells from undergoing mitosis bydisrupting or stabilizing microtubule polymerization, thus preventingformation of the mitotic spindle and subsequent cell division, resultingin inhibition of cancerous growth. Examples of microtubule-targetingagents are paclitaxel, docetaxel, vinblastine, vincristine, vinorelbine,duostatins, auristatins, maytansanoids, tubulysins, and dolastatin.

In one embodiment, the cytotoxic agent is auristatins or auristatinpeptide analogs and derivates ([131]; [132]). Auristatins have beenshown to interfere with microtubule dynamics, GTP hydrolysis and nuclearand cellular division [133] and have anti-cancer [134] and anti-fungalactivity [135]. The auristatin drug moiety may be attached to theantibody via a linker, through the N (amino) terminus or the C(terminus) of the peptidic drug moiety.

Exemplary auristatin embodiments include the N-terminus-linkedmonomethyl auristatin drug moieties DE and DF, disclosed in [136] anddescribed in [137].

In a particular embodiment, the cytotoxic agent is monomethyl auristatinE (MMAE);

wherein the antibody is linked to MMAE at the nitrogen (N) on theleft-hand side of the chemical structure above by the appropriatelinker.

In one embodiment, the cytotoxic agent monomethyl auristatin E (MMAE) islinked to the antibody via a valine-citrulline (VC) linker.

In another embodiment, the cytotoxic agent monomethyl auristatin E(MMAE) is linked to the antibody via a valine-citrulline (VC) linker andthe maleimidocaproyl (MC)linker, wherein the combination of thecytotoxic agent and the linkers has the chemical structure;

wherein MAb is the antibody.

In one embodiment, the cytotoxic agent is monomethyl auristatin F(MMAF);

wherein the antibody is linked to MMAF at the nitrogen (N) on theleft-hand side of the chemical structure above by the appropriatelinker.

In one embodiment, the cytotoxic agent monomethyl auristatin F (MMAF) islinked to the antibody via a maleimidocaproyl (mc)-linker, wherein thecombination of the cytotoxic agent and linker has the chemicalstructure;

wherein MAb is the antibody.

In one embodiment, the cytotoxic agent is duostatin3.

In another particular embodiment, the cytotoxic agent is a DNA-targetingagent.

The term “DNA-acting agent” as used herein, refers to a specific classof cytotoxic agents which are able to alkylate and/or cross-link DNA. Anexample of such a DNA-acting agent is IGN agents comprisingindolino-benzodiazepinedimers and pyrrolo[2,1-c][1,4]benzodiazepines(PBDs) which are highly potent by virtue of their ability to alkylateand cross-link DNA. Another example is IGN agents comprisingindolino-benzodiazepinemonomers which are highly potent by virtue of theability to alkylate only DNA. Duocarmycins are another class ofDNA-acting agents. Duocarmycins are small-molecule, synthetic DNA minorgroove binding alkylating agents. These compounds are suitable to targetsolid tumors as well as hematological tumors.

In one embodiment, the immunoconjugate comprises two to four cytotoxicmolecules per antibody. Depending on the chemical properties of thetoxin and the linker-toxin combination, two to four cytotoxic moleculesper antibody may be superior to more heavily loaded conjugates that arecleared more rapidly from the circulation than less loaded conjugates.The cytotoxic agent loading is represented by p and is the averagenumber of cytotoxic agent moieties per antibody in a molecule (alsodesignated as the drug to antibody ratio, DAR). The cytotoxic agentloading may range from 1 to 20 drug moieties per antibody and may occuron amino acids with useful functional groups such as, but not limitedto, amino or sulfhydryl groups, as in lysine or cysteine.

In one embodiment, the number of cytotoxic agents per antibody is from 1to 8, such as 2 to 7, such as 2 to 6, such as 2 to 5, such as 2 to 4,and such as 2 to 3.

In another embodiment, the immunoconjugate comprises four to eightcytotoxic molecules per antibody. In another embodiment, theimmunoconjugate comprises six to ten cytotoxic molecules per antibody.In yet another embodiment, the immunoconjugate comprises 10 to 30, suchas 15 to 25, such as 20, cytotoxic molecules per antibody.

Depending on the way of conjugation, p may be limited by the number ofattachment sites on the antibody, for example where the attachment is acysteine thiol or a lysine. Generally, antibodies do not contain manyfree and reactive cysteine thiol groups which may be linked to a drugmoiety as most cysteine thiol residues in antibodies exist as disulfidebridges. Therefore, in those embodiments, where the cytotoxic agent isconjugated via a cysteine thiol, the antibody may be reduced withreducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine(TCEP), under partial or fully reducing conditions, to generate reactivecysteine thiol groups. In certain embodiments, the drug loading for anADC of the invention ranges from 1 to about 8, as a maximum of 8 freecysteine thiol groups becomes available after (partial) reduction of theantibody (there are 8 cysteines involved in inter-chain disulfidebonding).

In one embodiment, the drug linker moiety is vcMMAE. The vcMMAE druglinker moiety and conjugation methods are disclosed in [27]; [28];[145], [146]; and [147], (which are incorporated herein by reference)vcMMAE is form by conjugation of the linker mc-vc-PAB and the cytotoxicmoiety MMAE, and the vcMMAE drug linker moiety is bound to the anti-AXLantibodies at the cysteine residues using a method similar to thosedisclosed therein.

In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF druglinker moiety and conjugation methods are disclosed in [138]; [139], and[140] (which are incorporated herein by reference), and the mcMMAF druglinker moiety is bound to the anti-AXL antibodies at the cysteineresidues using a method similar to those disclosed therein.

In one embodiment, the cytotoxic agent is linked to 1 or 2 lysineswithin the antibody amino acid sequence by K-Lock™ conjugation asdescribed in [58], [148], and [149], and the duostatin3 (also known asDuo3) is bound to the anti-AXL antibodies at the lysine residues using amethod similar to those described therein.

Other linker technologies may be used in the anti-AXL antibody drugconjugates of the invention, such as linkers comprising a hydroxylgroup.

In one embodiment, the linker is attached to free cysteine residues ofthe anti-AXL antibody obtained by (partial) reduction of the anti-AXLantibody.

In a particular embodiment, the linker is mc-vc-PAB and the cytotoxicagent is MMAE; or the linker SSP and the cytotoxic agent is DM1.

In a particular embodiment, the linker is MMC and the cytotoxic agent isDM1; or the linker is MC and the cytotoxic agent is MMAF.

In a particular embodiment, the linker is the cleavable linker AV1-Klock and the cytotoxic agent is duostatin3.

In one embodiment the immunoconjugate comprises the linker mc-vc-PAB,the cytotoxic agent MMAE and an antibody wherein the at least onebinding region comprises a VH region and a VL region selected from thegroup consisting of;

-   -   a) a VH region comprising SEQ ID No: 1 and a VL region        comprising SEQ ID No: 2 [107];    -   b) a VH region comprising SEQ ID No: 5 and a VL region        comprising SEQ ID No: 6 [148];    -   c) a VH region comprising SEQ ID No: 34 and a VL region        comprising SEQ ID No: 35 [733]    -   d) a VH region comprising SEQ ID No: 7 and a VL region        comprising SEQ ID No: 9 [154];    -   e) a VH region comprising SEQ ID No: 10 and a VL region        comprising SEQ ID No: 11 [171];    -   f) a VH region comprising SEQ ID No: 16 and a VL region        comprising SEQ ID No: 18 [183];    -   g) a VH region comprising SEQ ID No: 25 and a VL region        comprising SEQ ID No: 26 [613];    -   h) a VH region comprising SEQ ID No: 31 and a VL region        comprising SEQ ID No: 33 [726];    -   i) a VH region comprising SEQ ID No: 3 and a VL region        comprising SEQ ID No: 4 [140];    -   j) a VH region comprising SEQ ID No:8 and a VL region comprising        SEQ ID No:9 [154-M103L];    -   k) a VH region comprising SEQ ID No:12 and a VL region        comprising SEQ ID No:13 [172];    -   l) a VH region comprising SEQ ID No:14 and a VL region        comprising SEQ ID No:15 [181];    -   m) a VH region comprising SEQ ID No:17 and a VL region        comprising SEQ ID No:18 [183-N52Q];    -   n) a VH region comprising SEQ ID No:19 and a VL region        comprising SEQ ID No:20 [187];    -   o) a VH region comprising SEQ ID No:21 and a VL region        comprising SEQ ID No:22 [608-01];    -   p) a VH region comprising SEQ ID No:23 and a VL region        comprising SEQ ID No:24 [610-01];    -   q) a VH region comprising SEQ ID No:27 and a VL region        comprising SEQ ID No:28 [613-08];    -   r) a VH region comprising SEQ ID No:29 and a VL region        comprising SEQ ID No:30 [620-06]; and    -   s) a VH region comprising SEQ ID No:32 and a VL region        comprising SEQ ID No:33 [726-M101L].

In another alternative embodiment, an anti-AXL antibody drug conjugatedisclosed in the present invention comprises a conjugated nucleic acidor nucleic acid-associated molecule. In one such embodiment, theconjugated nucleic acid is a cytotoxic ribonuclease, an antisensenucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or animmunostimulatory nucleic acid (e.g., an immunostimulatory CpGmotif-containing DNA molecule).

In another alternative embodiment, an anti-AXL antibody of the inventionis conjugated to an aptamer or a ribozyme or a functional peptide analogor derivate thereof.

In another alternative embodiment, anti-AXL antibody drug conjugatescomprising one or more radiolabeled amino acids are provided. Aradiolabeled anti-AXL antibody may be used for both diagnostic andtherapeutic purposes (conjugation to radiolabeled molecules is anotherpossible feature). Non-limiting examples of labels for polypeptidesinclude ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, and ¹²⁵I, ¹³¹I, and ¹⁸⁶Re. Methodsfor preparing radiolabeled amino acids and related peptide derivativesare known in the art (see for instance [59] and [60], [61], [62], [63],[64] and [65]U.S. Pat. No. 5,697,902. For example, a radioisotope may beconjugated by a chloramine T method.

In one embodiment, the antibody is conjugated to a radioisotope or to aradioisotope-containing chelate. For example, the antibody can beconjugated to a chelator linker, e.g. DOTA, DTPA or tiuxetan, whichallows for the antibody to be complexed with a radioisotope. Theantibody may also or alternatively comprise or be conjugated to one ormore radiolabeled amino acids or other radiolabeled molecules. Aradiolabeled anti-AXL antibody may be used for both diagnostic andtherapeutic purposes. Non-limiting examples of radioisotopes include ³H,¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹²⁵I, ¹¹¹In, ¹³¹I, ¹⁸⁶Re, ²¹³Bs, ²²⁵Ac and²²⁷Th.

Anti-AXL antibodies may also be chemically modified by covalentconjugation to a polymer to for instance increase their circulatinghalf-life. Exemplary polymers, and methods to attach them to peptides,are illustrated in for instance [66]; [67]; [68]; and [69]. Additionalpolymers include polyoxyethylated polyols and polyethylene glycol (PEG)(e.g., a PEG with a molecular weight of between about 1,000 and about40,000, such as between about 2,000 and about 20,000). This may forexample be used if the anti-AXL antibody is a fragment.

Any method known in the art for conjugating the anti-AXL antibodyaccording to the present invention to the conjugated molecule(s), suchas those described above, may be employed, including the methodsdescribed by [70], [71] and [72]. Such antibodies may be produced bychemically conjugating the other moiety to the N-terminal side orC-terminal side of the anti-AXL antibody (e.g., an anti-AXL antibody Hor L chain) (see, e.g., [73]). Such conjugated antibody derivatives mayalso be generated by conjugation at internal residues or sugars, ornon-naturally occurring amino acids or additional amino acids that havebeen introduced into the antibody constant domain, where appropriate.

The agents may be coupled either directly or indirectly to an anti-AXLantibody disclosed in the present invention. One example of indirectcoupling of a second agent is coupling via a spacer moiety to cysteineor lysine residues in the antibody. In one embodiment, an anti-AXLantibody is conjugated, via a spacer or linker, to a prodrug moleculethat can be activated in vivo to a therapeutic drug. Afteradministration, the spacers or linkers are cleaved by tumorcell-associated enzymes or other tumor-specific conditions, by which theactive drug is formed. Examples of such pro-drug technologies andlinkers are described in [74], [75], [76], [77], [78] and [79] (allincorporated herein by reference) Suitable antibody-pro-drug technologyand duocarmycin analogs can also be found in [80] (incorporated hereinby reference).

In one embodiment, the anti-AXL antibody of the present invention isattached to a chelator linker, e.g. tiuxetan, which allows for theantibody to be conjugated to a radioisotope.

Compositions

In a further aspect, the invention relates to a composition comprisingthe antibody, the bispecific antibody, or the immunoconjugate of theinvention.

In another aspect, the invention relates to a pharmaceutical compositioncomprising the antibody, bispecific or immunoconjugate according to theinvention and a pharmaceutical carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in [81].

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the antibodyor antibody conjugate of the present invention and the chosen mode ofadministration. Suitability for carriers and other components ofpharmaceutical compositions is determined based on the lack ofsignificant negative impact on the desired biological properties of thechosen compound or pharmaceutical composition of the present invention(e.g., less than a substantial impact (10% or less relative inhibition,5% or less relative inhibition, etc.) upon antigen binding).

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e. g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition.

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the amide thereof, the route of administration,the time of administration, the rate of excretion of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompositions employed, the age, sex, weight, condition, general healthand prior medical history of the patient being treated, and like factorswell known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and may beselected by those of ordinary skill in the art.

In one embodiment, the pharmaceutical composition of the presentinvention is administered parenterally.

The terms “parenteral administration” and “administered parenterally” asused herein refers to modes of administration other than enteral andtopical administration, usually by injection, and include epidermal,intravenous, intramuscular, intra-arterial, intrathecal, intracapsular,intra-orbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment, the pharmaceutical composition of the presentinvention is administered by intravenous or subcutaneous injection orinfusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption-delaying agents,and the like that are physiologically compatible with a compound of thepresent invention.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate-buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion.

The use of such media and agents for pharmaceutically active substancesis known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thecompounds of the present invention may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicro-encapsulated delivery systems. Such carriers may include gelatin,glyceryl monostearate, glyceryl distearate, biodegradable, biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, poly-ortho-esters, and polylactic acid alone or with awax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See e.g., [82].

In one embodiment, the compounds of the present invention may beformulated to ensure proper distribution in vivo. Pharmaceuticallyacceptable carriers for parenteral administration include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.Other active or therapeutic compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, micro-emulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe an aqueous or a non-aqueous solvent or dispersion medium containingfor instance water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), and suitable mixturesthereof, vegetable oils, such as olive oil, and injectable organicesters, such as ethyl oleate. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin. Sterile injectable solutions may be prepared byincorporating the active compound in the required amount in anappropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum-drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum-drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The pharmaceutical composition of the present invention may contain oneantibody, bispecific antibody or ADC of the present invention, acombination of an antibody, a bispecific antibody or ADC according tothe invention with another therapeutic compound, or a combination ofcompounds of the present invention.

Nucleic Acid Constructs, Expression Vectors, and Host Cells

In one aspect, the present invention relates to a nucleic acid constructencoding one or more sequences set out in Table 1. Thus, the presentinvention relates to nucleic acid constructs encoding any one of thesequences set out in SEQ ID Nos.: 1 to 135. In one embodiment, thenucleic acid construct encodes at least one of the amino acid sequenceselected from the group consisting of SEQ ID Nos.: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, and 135. Thus, in oneembodiment, the nucleic acid construct encodes an antibody according toany aspect or embodiment herein described.

In a particular embodiment, the nucleic acid construct encodes at leastone of the amino acid sequences selected from the group consisting ofSEQ ID Nos.: 46, 47, 48, 49, 50, 36, 37, 38, 39, 40, 114, 115, 116, 117,and 118.

In a further aspect, the invention relates to an expression vectorencoding an antibody of the invention. Thus, the expression vectorcomprises one or more nucleic acid constructs according to any aspect orembodiment herein described. Such expression vectors may in oneembodiment be used to express the anti-AXL antibody of the presentinvention. The expressed anti-AXL antibody may subsequently beconjugated to a moiety as described herein. In another embodiment theanti-AXL antibody may subsequently be used to generate a bispecificantibody as described herein.

In one embodiment, the expression vector of the invention comprises anucleic acid sequence encoding one or more of the heavy chain (VH) CDR3amino acid sequences selected from the group consisting of: SEQ ID Nos.:38, 43, 48, 53, 54, 59, 64, 69, 75, 80, 85, 90, 95, 100, 105, 110, 111,116, 120, 122, 125, and 127.

In a particular embodiment, the expression vector of the inventioncomprises a nucleic acid sequence encoding one or more of the VH CDR1,CDR2, and CDR3 amino acid sequences selected from the group consistingof: SEQ ID Nos.: 36-38, 41-43, 46-48, 51-54, 57-59, 62-64, 67-69, 72-75,78-80, 83-85, 88-90, 93-95, 98-100, 103-105, 108-110, and 114-116.

In one embodiment, the expression vector of the invention comprises anucleic acid sequence encoding one or more of the light chain (VL) CDR3amino acid sequences selected from the group consisting of: SEQ ID Nos.:40, 45, 50, 56, 61, 66, 71, 77, 82, 87, 92, 97, 102, 107, 113, and 118.

In another particular embodiment, the expression vector of the inventioncomprises a nucleic acid sequence encoding one or more of the VH aminoacid sequences selected from the group consisting of: SEQ ID Nos.: 1, 3,5, 7, 8, 10, 12, 14, 16, 17, 19, 21, 23, 25, 27, 29, 31, 32, and 34.

In another particular embodiment, the expression vector of the inventioncomprises a nucleic acid sequence encoding one or more of the VL aminoacid sequences selected from the group consisting of: SEQ ID Nos.: 2, 4,6, 9, 11, 13, 15, 18, 20, 22, 24, 26, 28, 30, 33, and 35.

In one embodiment, the expression vector of the invention comprises anucleic acid sequence encoding one or more of the amino acid sequencesselected from the group consisting of: SEQ ID Nos.: 1 to 35.

In a particular embodiment, the expression vector of the inventioncomprises a nucleic acid sequence encoding variants of one or more ofthe above amino acid sequences, said variants having at most 25 aminoacid modifications, such as 20, such as at most 15, 14, 13, 12, or 11amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid modifications, such as deletions or insertions, preferablysubstitutions, such as conservative or non-conservative substitutions,or at least 80% identity to any of said sequences, such as at least 85%identity or 90% identity or 95% identity, such as 96% identity or 97%identity or 98% identity or 99% identity to any of the aforementionedamino acid sequences. The present invention also relates to nucleic acidsequences different from the above mentioned nucleic acid sequences butwhich due to the variance of the genetic code encode the same amino acidsequence as an antibody of the present invention. E.g. the nucleic acidsequence may vary but result in an identical amino acid sequence as anyamino acid sequence herein described. It is well-known for the skilledperson how to identify such further nucleic acid sequences based on thegenetic code.

In a further embodiment, the expression vector further comprises anucleic acid sequence encoding the constant region of a light chain, aheavy chain or both light and heavy chains of an antibody, e.g. a humanIgG1, K monoclonal antibody.

Such expression vectors as described above may be used for recombinantproduction of antibodies of the invention.

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ananti-AXL antibody-encoding nucleic acid is comprised in a naked DNA orRNA vector, including, for example, a linear expression element (asdescribed in for instance [83]), a compacted nucleic acid vector (asdescribed in for instance [84] and/or [85]), a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleicacid vector (as described in for instance [86]), or as a precipitatednucleic acid vector construct, such as a CaP04-precipitated construct(as described in for instance [87], [88], [89], and [90]). Such nucleicacid vectors and the usage thereof are well known in the art (see forinstance [91] and [92]).

In one embodiment, the vector is suitable for expression of the anti-AXLantibody in a bacterial cell. Examples of such vectors includeexpression vectors such as BlueScript (Stratagene), pIN vectors ([93],pET vectors (Novagen, Madison Wis.) and the like).

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: [94], and [95]).

A nucleic acid construct and/or vector may also comprises a nucleic acidsequence encoding a secretion/localization sequence, which can target apolypeptide, such as a nascent polypeptide chain, to the periplasmicspace or into cell culture media. Such sequences are known in the art,and include secretion leader or signal peptides, organelle targetingsequences (e. g., nuclear localization sequences, ER retention signals,mitochondrial transit sequences, chloroplast transit sequences),membrane localization/anchor sequences (e. g., stop transfer sequences,GPI anchor sequences), and the like.

In an expression vector of the invention, anti-AXL antibody-encodingnucleic acids may comprise or be associated with any suitable promoter,enhancer, and other expression-facilitating elements. Examples of suchelements include strong expression promoters (e. g., human CMV IEpromoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE (the skilled artisanwill recognize that such terms are actually descriptors of a degree ofgene expression under certain conditions).

In one embodiment, the anti-AXL-antibody-encoding expression vector maybe positioned in and/or delivered to the host cell or host animal via aviral vector.

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an anti-AXL antibody of the invention as defined herein or abispecific molecule of the invention as defined herein. Examples of hostcells include yeast, bacterial and mammalian cells, such as CHO or HEKcells or derivatives thereof. For example, in one embodiment, thepresent invention provides a cell comprising a nucleic acid stablyintegrated into the cellular genome that comprises a sequence coding forexpression of an anti-AXL antibody of the present invention. In anotherembodiment, the present invention provides a cell comprising anon-integrated nucleic acid, such as a plasmid, cosmid, phagemid, orlinear expression element, which comprises a sequence coding forexpression of an anti-AXL antibody of the invention.

In a further aspect, the present invention relates to a host cellcomprising a vector according to any aspect and embodiments hereindescribed. In one embodiment, the anti-AXL antibodies described hereinare provided by use of recombinant eukaryotic, prokaryotic or microbialhost cell which produces the antibody. Accordingly, the inventionprovides a recombinant host cell, such as a recombinant prokaryotic,recombinant eukaryotic, or recombinant microbial host cell. Examples ofhost cells include yeast, bacterial and mammalian cells, such as CHO orHEK-293 cells. For example, in one embodiment, the host cell comprises anucleic acid stably integrated into the cellular genome that comprises asequence coding for expression of an anti-AXL antibody described herein.In one embodiment, the host cell comprises a nucleic acid stablyintegrated into the cellular genome that comprise a sequence coding forexpression of a first or a second polypeptide described herein. Inanother embodiment, the host cell comprises a non-integrated nucleicacid, such as a plasmid, cosmid, phagemid, or linear expression element,which comprises a sequence coding for expression of an anti-AXLantibody, a first or a second polypeptide described herein.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which an expression vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell, but also tothe progeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, transfectomas,such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocyticcells, and prokaryotic cells such as E. coli and other eukaryotic hostssuch as plant cells and fungi.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing the antibody or a target antigen, such as CHOcells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungi,including yeast cells.

In another aspect, the present invention relates to a hybridoma whichproduces an antibody according to any aspect or embodiment hereindescribed. Thus, an antibody may be obtained from hybridomas preparedfrom murine splenic B cells obtained from mice immunized with an antigenof interest, for instance in form of cells expressing the antigen on thesurface, or a nucleic acid encoding an antigen of interest. Monoclonalantibodies may also be obtained from hybridomas derived fromantibody-expressing cells of immunized humans or non-human mammals suchas rabbits, rats, dogs, primates, etc.

Human antibodies may be generated using transgenic or transchromosomalmice, e.g. HuMAb mice, carrying parts of the human immune system ratherthan the mouse system. The HuMAb mouse contains a human immunoglobulingene minilocus that encodes unrearranged human heavy (μ and γ) and κlight chain immunoglobulin sequences, together with targeted mutationsthat inactivate the endogenous μ and κ chain loci [96]. Accordingly, themice exhibit reduced expression of mouse IgM or κ and in response toimmunization, the introduced human heavy and light chain transgenes,undergo class switching and somatic mutation to generate high affinityhuman IgG,κ monoclonal antibodies [96], [97], [98] and [99]. Thepreparation of HuMAb mice is described in detail in [100], [101], [102],[103], and [104]. See also [105] to [121]. Splenocytes from thesetransgenic mice may be used to generate hybridomas that secrete humanmonoclonal antibodies according to well-known techniques.

Further, human antibodies of the present invention or antibodies of thepresent invention from other species may be identified throughdisplay-type technologies, including, without limitation, phage display,retroviral display, ribosomal display, mammalian display, yeast displayand other techniques known in the art, and the resulting molecules maybe subjected to additional maturation, such as affinity maturation, assuch techniques are well known in the art.

Thus, in one aspect, the present invention relates to a method forproducing an antibody according to any aspect or embodiment hereindescribed, comprising the steps

-   -   a) culturing a host cell or hybridoma according to any aspect or        embodiment herein described; and    -   b) purifying the antibody from the culture media.        Therapeutic Applications

In another aspect, the invention relates to the antibody, bispecificantibody, or immunoconjugate or ADC of the present invention, as definedin any aspect or embodiment herein, for use as a medicament.

The anti-AXL antibodies of the present invention can be used in thetreatment or prevention of disorders involving cells expressing AXL. Forexample, the antibodies may be administered to cells in culture, e.g.,in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat orprevent disorders involving AXL-expressing cells. As used herein, theterm “subject” is typically a human to whom the anti-AXL antibody or ADCis administered. Subjects may for instance include human patients havingdisorders that may be corrected or ameliorated by modulating AXLfunction or by killing of the AXL-expressing cell, directly orindirectly.

In one aspect, the present invention relates to the antibody, bispecificantibody, or immunoconjugate of the present invention, as defined in anyaspect or embodiment herein, for use in the treatment of cancer.

In one aspect, the invention provides a method for modulatingAXL-associated signaling in an AXL-expressing cell by contacting thecell with an anti-AXL antibody or ADC according to any aspect orembodiment herein described.

In one embodiment, the invention provides a method for killing anAXL-expressing cell by contacting the cell with an anti-AXL antibody orADC of the invention. Without being limited to theory, antibody-mediatedor ADC-mediated crosslinking or clustering (e.g., due to the Fc-regionof AXL-bound antibodies binding to FcR-expressing cells) of AXLmolecules on the surface of a cell can lead to apoptosis of the cell.

In one embodiment, the invention provides a method for killing anAXL-expressing cell by contacting the cell with an AXL-specific antibodyor ADC of the invention in the presence of effector cells capable ofinducing an Fc-dependent cellular response such as ADCC or ADCP. In thisembodiment, the antibody is typically full-length and of an isotypeleading to an ADCC or ADCP response, such as, e.g., an IgG1,κ isotype.

In one embodiment, the invention provides a method for killing anAXL-expressing cell by contacting the cell with an AXL-specific antibodyor ADC of the invention in the presence of complement proteins, such ascomplement proteins present in normal human serum, that may be activatedand thereby inducing CDC after binding of AXL-specific antibody or ADCto the plasma membrane of AXL-expressing cells. In this embodiment, theantibody is typically full-length and of an isotype capable of inducingactivation of the complement system, such as, e.g., an IgG1,κ isotype.

The anti-AXL antibodies of the invention may be characterized byinternalization upon binding to AXL, making them suitable for an ADCapproach using an ADC as described in any aspect or embodiment describedherein.

Accordingly, in one embodiment, the invention provides a method forkilling an AXL-expressing cell by contacting the cell with an ADC of theinvention which requires internalization and trafficking to lysosomesfor specific (i.e. cleavable linker) or non-specific (non-cleavablelinker) proteolytic cleavage of the anti-AXL antibody-linker-drugcomplex.

In one embodiment, the present invention relates to a method forinterfering with AXL-mediated regulation of the innate or adaptiveimmune response, such as by binding of an AXL-specific antibody or ADCaccording to the invention to AXL-expressing macrophages, dendriticcells or NK cells.

In another embodiment, the invention provides for a method of killing anAXL-expressing cell by contacting the cell with an ADC of the inventionwherein the anti-AXL antibody is linked to a therapeutic moiety via alinker allowing for release of the drug once the ADC is internalized,e.g., by a change in pH or reducing conditions. Suitable linkertechnology is known in the art, as described above.

In another aspect, the present invention provides methods for treatingor preventing a disorder involving cells expressing AXL in a subject,which method comprises administration of a therapeutically effectiveamount of an anti-AXL antibody, bispecific antibody or ADC of thepresent invention to a subject in need thereof. The method typicallyinvolves administering to a subject an anti-AXL antibody, a bispecificantibody or ADC according to the present invention in an amounteffective to treat or prevent the disorder.

In a particular aspect, an anti-AXL antibody or ADC is administeredprophylactically in order to reduce the risk of developing cancer, delaythe onset of an event in cancer progression or reduce the risk ofrecurrence when a cancer is in remission and/or a primary tumor has beensurgically removed. In the latter case, the anti-AXL antibody could, forexample, be administered in association with (i.e., before, during, orafter) the surgery. Prophylactic administration may also be useful inpatients wherein it is difficult to locate a tumor that is believed tobe present due to other biological factors.

Cells with high AXL expression, such as over-expression or aberrantexpression of AXL, as found in some cancer cells, are particularly goodtargets for the anti-AXL antibodies, bispecific antibodies or ADCs ofthe invention, since more antibodies or ADCs may be bound per tumorcell. Tissues that heterogeneously express AXL such as tumor tissue mayalso be a suitable target for the anti-AXL antibodies, bispecificantibodies, ADCs or anti-AXL-ADCs of the invention. Thus, in one aspect,the disorder involving cells expressing AXL is cancer, i.e., atumorigenic disorder, such as a disorder characterized by the presenceof tumor cells expressing AXL including, for example, disorders wherethe cells are from a solid tumor or hematological tumor. AXL expressionhas been described in, e.g., non-small-cell lung cancer (NSCLC; [122]),pancreatic cancer [123], esophageal cancer [124], endometrial cancer[125].

Exemplary cells expressing AXL thus include cancer cells such as, e.g.,cells from non-small cell lung cancer, pancreatic cancer and esophagealcancer.

In one aspect, the present invention provides methods for treating orpreventing cancer comprising administering the antibody, the bispecificantibody, the immunoconjugate, the composition, or the pharmaceuticalcomposition according to the present invention to a subject in needthereof.

In one embodiment, the cancer is a solid tumor expressing AXL or anAXL-expressing hematological cancer. In one embodiment, thehematological cancer is Acute Myeloid Leukemia (AML). In one embodiment,the solid tumor expressing AXL is lung cancer or epidermoid carcinoma.

Thus, the present invention relates to methods comprising administrationof a therapeutically effective amount of an anti-AXL antibody or ADC ofthe present invention to a subject in need thereof.

In one aspect, the present invention relates to a method for inhibitinggrowth and/or proliferation of a tumor cell expressing AXL, comprisingadministration, to an individual in need thereof, of an antibody,bispecific antibody, immunoconjugate, composition, or pharmaceuticalcomposition according to any aspect or embodiment herein described.

In one aspect, the present invention relates to a method for inhibitingmigration and/or invasion of a tumor cell expressing AXL, comprisingadministration, to an individual in need thereof, of an antibody,bispecific antibody, immunoconjugate, composition, or pharmaceuticalcomposition according to any aspect or embodiment herein described.

In one aspect, the present invention relates to a method for inhibitingresistance to targeted therapy, such as EGFR- or BRAF-targeted therapy,or to chemotherapeutic agents, comprising administration, to anindividual in need thereof, of an antibody, bispecific antibody,immunoconjugate, composition, or pharmaceutical composition according toany aspect or embodiment herein described.

In one aspect, the present invention relates to a method for targetingor inhibiting tumor-associated macrophages, comprising administration,to an individual in need thereof, of an antibody, bispecific antibody,immunoconjugate, composition, or pharmaceutical composition according toany aspect or embodiment herein described.

In one aspect, the present invention provides methods for treating orpreventing a solid tumor, which method comprises administration of atherapeutically effective amount of an anti-AXL antibody or ADC of thepresent invention to a subject in need thereof, and wherein the solidtumor is a melanoma, carcinoma, sarcoma, adenoma and/or a glioma. In oneembodiment, the cancer is selected from the group consisting ofendometrial/cervical cancer, lung cancer (such as, e.g., small cell lungcancer or non-small cell lung cancer), thyroid cancer, colon cancer,kidney cancer, ovary cancer, breast cancer, esophagus cancer, skincancer, malignant melanoma and pancreatic cancer.

In one embodiment, the cancer is pancreatic cancer, such as unresectableadvanced or metastatic pancreatic cancer. In other separate and specificembodiments, the cancer is endometrial/cervical cancer, or lung cancer.In one embodiment the cancer is a thyroid cancer. In one embodiment thecancer is a colon cancer. In one embodiment the cancer is a kidneycancer. In one embodiment the cancer is ovarian cancer. In oneembodiment the cancer is breast cancer such as estrogen receptor alphanegative cancer or estrogen receptor alpha positive. In one embodimentthe cancer is triple negative breast cancer (i.e. breast cancer testednegative for estrogen receptors (ER−), progesterone receptors (PR−), andhuman epidermal growth factor receptor 2 (HER2−). In one embodiment thecancer is esophagus cancer. In one embodiment the cancer is skin cancer.In one embodiment the cancer is melanoma such as malignant melanoma. Inone embodiment the cancer is Acute Myeloid Leukemia (AML). In oneembodiment the cancer is resistant to chemotherapy, thyrosine kinaseinhibitors and or BRAF inhibitors. In one embodiment the cancer isresistant to EGFR targeted therapy.

In one aspect, the present invention relates to an antibody binding tothe extracellular domain of AXL such as the Ig1-like domain of AXL, suchas the Ig2-like domain of AXL, such as the FN1 domain of AXL, or such asthe FN2 domain of AXL, wherein said antibody is for use as a medicament.

In a particular embodiment the antibody comprises at least one bindingregion comprising a VH region and a VL region, wherein the VH regioncomprises the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and38, and the VL region comprises the sequences of SEQ ID Nos.: 39, GAS,and 40, wherein said antibody is for use as a medicament.

In a particular embodiment the antibody comprises at least one bindingregion comprising a VH region and a VL region, wherein the VH regioncomprises the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and38, and the VL region comprises the sequences of SEQ ID Nos.: 39, GAS,and 40, wherein said antibody is for use in treatment or prevention ofcancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of thyroidcancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of coloncancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of kidneycancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of ovariancancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of breastcancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of triplenegative breast cancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of esophaguscancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ

ID Nos.: 36, 37 and 38, and the VL region comprises the sequences of SEQID Nos.: 39, GAS, and 40, wherein said antibody is for use in treatmentor prevention of skin cancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of melanomasuch as malignant melanoma.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of lungcancer (such as, e.g., small cell lung cancer or non-small cell lungcancer (NSCLC)).

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and theVL region comprises the sequences of SEQ ID Nos.: 39, GAS, and 40,wherein said antibody is for use in treatment or prevention of AcuteMyeloid Leukemia (AML).

Hereby embodiments are provided for the treatment or prevention ofcancer. Said embodiments may be coupled or linked to a cytotoxic agentto increase the efficacy or effect of the treatment.

In a particular aspect of the present invention the antibodies disclosedherein may be linked to a cytotoxic agent such as an auristatin (suchas, e.g monomethyl auristatin E (MMAE)) to form an immunoconjugate foruse as a medicament, such as e.g. for the treatment or prevention ofcancer.

In one aspect the present invention relates to an immunoconjugatecomprising an antibody which binds to AXL, wherein the antibody does notcompete for AXL binding with the ligand Gas6.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use as a medicament.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of cancer. In one embodiment thereof said antibody is linkedto monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of thyroid cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of colon cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of kidney cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of ovarian cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of breast cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of triple negative breast cancer. In one embodiment thereofsaid antibody is linked to monomethyl auristatin E by the linkermc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of lung cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of esophagus cancer. In one embodiment thereof said antibodyis linked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of melanoma such as malignant melanoma. In one embodimentthereof said antibody is linked to monomethyl auristatin E by the linkermc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of lung cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 36, 37 and 38, and the VL regioncomprises the sequences of SEQ ID Nos.: 39, GAS, and 40, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of Acute Myeloid Leukemia. In one embodiment thereof saidantibody is linked to monomethyl auristatin E by the linker mc-vc-PAB.

Hereby embodiments are provided wherein the antibody is linked to acytotoxic agent such as an auristatin to form an immunoconjugate. Theantibody and the cytotoxic agent may be linked by amaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PAB)linker.

In an additional aspect of the invention, the invention relates toantibodies that bind to the extracellular domain of AXL such as theIg2-like domain of AXL, wherein said antibody is for use as amedicament.

In a particular embodiment the antibody comprises at least one bindingregion comprising a VH region and a VL region, wherein the VH regioncomprises the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and48, and the VL region comprises the sequences of SEQ ID Nos.: 49, AAS,and 50, wherein said antibody is for use as a medicament.

In a particular embodiment the antibody comprises at least one bindingregion comprising a VH region and a VL region, wherein the VH regioncomprises the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and48, and the VL region comprises the sequences of SEQ ID Nos.: 49, AAS,and 50, wherein said antibody is for use in treatment or prevention ofcancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of thyroidcancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of coloncancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of kidneycancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of ovariancancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of breastcancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of triplenegative breast cancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of esophaguscancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of skincancer.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of melanomasuch as malignant melanoma.

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50wherein said antibody is for use in treatment or prevention of lungcancer (such as, e.g., small cell lung cancer or non-small cell lungcancer).

In on embodiment the antibody comprises at least one binding regioncomprising a VH region and a VL region, wherein the VH region comprisesthe CDR1, CDR2 and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and theVL region comprises the sequences of SEQ ID Nos.: 49, AAS, and 50,wherein said antibody is for use in treatment or prevention of AcuteMyeloid Leukemia (AML).

Hereby embodiments are provided for the treatment or prevention ofcancer. Said embodiments may be coupled or linked with a cytotoxic agentto increase the efficacy or effect of the treatment.

In a particular aspect of the present invention the antibodies disclosedherein may be linked with a cytotoxic agent such as an auristatin (suchas, e.g monomethyl auristatin E (MMAE)) to form an immunoconjugate foruse as a medicament, such as e.g. for the treatment or prevention ofcancer.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use as a medicament.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for in treatment orprevention of cancer.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of thyroid cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of colon cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of kidney cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of ovarian cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of breast cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of triple negative breast cancer. In one embodiment thereofsaid antibody is linked to monomethyl auristatin E by the linkermc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of lung cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of esophagus cancer. In one embodiment thereof said antibodyis linked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of melanoma such as malignant melanoma. In one embodimentthereof said antibody is linked to monomethyl auristatin E by the linkermc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50, wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of lung cancer. In one embodiment thereof said antibody islinked to monomethyl auristatin E by the linker mc-vc-PAB.

In one embodiment the invention relates to an immunoconjugate comprisingan antibody comprising at least one binding region comprising a VHregion and a VL region, wherein the VH region comprises the CDR1, CDR2and CDR3 sequences of SEQ ID Nos.: 46, 47 and 48, and the VL regioncomprises the sequences of SEQ ID Nos.: 49, AAS, and 50 wherein saidantibody is linked to monomethyl auristatin E to form animmunoconjugate, and said immunoconjugate is for use in treatment orprevention of Acute Myeloid Leukemia. In one embodiment thereof saidantibody is linked to monomethyl auristatin E by the linker mc-vc-PAB.

Hereby embodiments are provided wherein the antibody is linked to acytotoxic agent such as an auristatin to form an immunoconjugate. Theantibody and the cytotoxic agent may be linked by amaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PAB)linker.

In an embodiment selection of patients to be treated with anti-AXLantibodies is based on the level of AXL expression in a sample, such asa sample containing tumor cells, or by detecting AXL-expressing tumorsusing labeled anti-AXL antibodies or antibody fragments, e.g., those ofthe invention. Exemplary diagnostic assays for determiningAXL-expression using AXL antibodies of the invention are describedherein. The efficient dosages and dosage regimens for the anti-AXLantibody or ADC depend on the disease or condition to be treated and maybe determined by the persons skilled in the art.

A physician having ordinary skill in the art may readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. In relation hereto, when referring to a pharmaceuticalcomposition it is to be understood also to comprise a composition assuch, or vice versa. For example, the physician could start doses of theanti-AXL antibody employed in the pharmaceutical composition at levelslower than that required in order to achieve the desired therapeuticeffect and gradually increase the dosage until the desired effect isachieved. In general, a suitable dose of a pharmaceutical composition ofthe present invention will be that amount of the compound which is thelowest dose effective to produce a therapeutic effect according to aparticular dosage regimen. Such an effective dose will generally dependupon the factors described above.

For example, an “effective amount” for therapeutic use may be measuredby its ability to stabilize the progression of disease. The ability of acompound to inhibit cancer may, for example, be evaluated in an animalmodel system predictive of efficacy in human tumors. Alternatively, thisproperty of a composition may be evaluated by examining the ability ofthe compound to inhibit cell growth or to induce cytotoxicity by invitro assays known to the skilled practitioner. A therapeuticallyeffective amount of a therapeutic compound may decrease tumor size, orotherwise ameliorate symptoms in a subject. One of ordinary skill in theart would be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

An exemplary, non-limiting range for a therapeutically effective amountof an anti-AXL antibody of the present invention is about 0.1-100 mg/kg,such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such asabout 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1,about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.

An exemplary, non-limiting range for a therapeutically effective amountof an anti-AXL ADC of the invention is 0.02-100 mg/kg, such as about0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for exampleabout 0.5-2 mg/kg.

Administration may e.g. be intravenous, intramuscular, intraperitoneal,or subcutaneous, and for instance administered proximal to the site ofthe target.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation.

In one embodiment, the efficacy-safety window is optimized by loweringspecific toxicity such as for example by lowering the drug-antibodyratio (DAR) and/or mixing of anti-AXL ADC with unlabeled anti-AXLantibody.

In one embodiment, the efficacy of the treatment is monitored during thetherapy, e.g. at predefined points in time. In one embodiment, theefficacy may be monitored by measuring the level of AXL in a samplecontaining tumor cells, by visualization of the disease area, or byother diagnostic methods described further herein, e.g. by performingone or more PET-CT scans, for example using a labeled anti-AXL antibody,fragment or mini-antibody derived from the AXL-specific antibody of thepresent invention.

If desired, an effective daily dose of a pharmaceutical composition maybe administered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In another embodiment, the anti-AXLantibodies are administered by slow continuous infusion over a longperiod, such as more than 24 hours, in order to minimize any unwantedside effects.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition as described above.

An effective dose of an anti-AXL antibody, bispecific antibody or ADC ofthe invention may also be administered using a weekly, biweekly ortriweekly dosing period. The dosing period may be restricted to, e.g., 8weeks, 12 weeks or until clinical progression has been established.

For example, in one embodiment, the anti-AXL antibody, bispecificantibody or ADC is administered by infusion in a weekly dosage ofbetween 10 and 500 mg/m², such as between 200 and 400 mg/m². Suchadministration may be repeated, e.g., 1 to 8 times, such as 3 to 5times. The administration may be performed by continuous infusion over aperiod of from 1 to 24 hours, such as of from 1 to 12 hours.

In another embodiment, the anti-AXL antibody, bispecific antibody or ADCis administered by infusion every three weeks in a dosage of between 10and 500 mg/m², such as between 50-200 mg/m². Such administration may berepeated, e.g., 1 to 8 times, such as 3 to 5 times. The administrationmay be performed by continuous infusion over a period of from 1 to 24hours, such as of from 1 to 12 hours.

In one embodiment, an anti-AXL ADC is administered as a single dose ofabout 0.1-10 mg/kg, such as about 1-3 mg/kg, every week or every thirdweek for up to twelve times, up to eight times, or until clinicalprogression. The administration may be performed by continuous infusionover a period of from 1 to 24 hours, such as of from 1 to 12 hours. Suchregimens may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the anti-AXL antibodies of the present invention.

In one embodiment, the anti-AXL antibodies are administered asmaintenance therapy, such as, e.g., once a week for a period of sixmonths or more.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of a compound of the present inventionin an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1,1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively,at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 after initiation of treatment, or any combinationthereof, using single or divided doses every 24, 12, 8, 6, 4, or 2hours, or any combination thereof.

Parenteral compositions may be formulated in dosage unit form for easeof administration and uniformity of dosage. Dosage unit form as usedherein refers to physically discrete units suited as unitary dosages forthe subjects to be treated; each unit contains a predetermined quantityof active compound calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

Diagnostic Applications

The anti-AXL antibodies of the invention may also be used for diagnosticpurposes, using a composition comprising an anti-AXL antibody asdescribed herein. Accordingly, the invention provides diagnostic methodsand compositions using the anti-AXL antibodies described herein. Suchmethods and compositions can be used for purely diagnostic purposes,such as detecting or identifying a disease involving AXL-expressingcells, as well as for monitoring of the progress of therapeutictreatments, monitoring disease progression, assessing status aftertreatment, monitoring for recurrence of disease, evaluating risk ofdeveloping a disease, and the like.

In one aspect, the anti-AXL antibodies of the present invention are usedex vivo, such as in diagnosing a disease in which cells expressing AXLare indicative of disease or involved in the pathogenesis, by detectinglevels of AXL or levels of cells which express AXL on their cell surfacein a sample taken from a patient. This may be achieved, for example, bycontacting the sample to be tested, optionally along with a controlsample, with the anti-AXL antibody under conditions that allow forbinding of the antibody to AXL. Complex formation can then be detected(e.g., using an ELISA). When using a control sample along with the testsample, the level of anti-AXL antibody or anti-AXL antibody-AXL complexis analyzed in both samples and a statistically significant higher levelof anti-AXL antibody or anti-AXL antibody-AXL complex in the test sampleindicates a higher level of AXL in the test sample compared with thecontrol sample.

Examples of conventional immunoassays in which anti-AXL antibodies ofthe present invention can be used include, without limitation, ELISA,RIA, FACS assays, plasmon resonance assays, chromatographic assays,tissue immunohistochemistry, Western blot, and/or immunoprecipitation.

Accordingly, in one embodiment, the present invention relates to amethod of diagnosing a disease characterized by involvement oraccumulation of AXL-expressing cells, comprising administering anantibody, bispecific antibody, immunoconjugate, composition orpharmaceutical composition according to any aspect or embodiment hereindescribed, to a subject, optionally wherein the antibody is labeled witha detectable label, and wherein the amount of AXL-expressing cellscorrelates with or is indicative of disease.

In one embodiment, the invention relates to a method for detecting thepresence of AXL antigen, or a cell expressing AXL, in a samplecomprising:

contacting the sample with an anti-AXL antibody of the invention underconditions that allow for binding of the anti-AXL antibody to AXL in thesample; and

analyzing whether a complex has been formed. Typically, the sample is abiological sample. The term “AXL antigen” as used in this context,refers both soluble and cell bound AXL antigen.

In one embodiment, the sample is a tissue sample known or suspected ofcontaining AXL antigen and/or cells expressing AXL. For example, in situdetection of AXL expression may be accomplished by removing ahistological specimen from a patient, and providing the antibody of thepresent invention to such a specimen. The antibody may be provided byapplying or by overlaying the antibody to the specimen, which is thendetected using suitable means. It is then possible to determine not onlythe presence of AXL or AXL-expressing cells, but also the distributionof AXL or AXL-expressing cells in the examined tissue (e.g., in thecontext of assessing the spread of cancer cells). Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) maybe modified in order to achieve such in situ detection.

In the above assays, the anti-AXL antibody can be labeled with adetectable substance to allow AXL-bound antibody to be detected.Alternatively, bound (primary) anti-AXL antibody may be detected by asecondary antibody which is labeled with a detectable substance andwhich binds to the primary antibody. Furthermore, in the above assays, adiagnostic composition comprising an antibody or bispecific antibodyaccording to any aspect or embodiments herein described may be used.Thus, in one aspect, the present invention relates to a diagnosticcomposition comprising an antibody or bispecific antibody according toany aspect or embodiment herein described.

The level of AXL in a sample can also be estimated by a competitionimmunoassay utilizing AXL standards labeled with a detectable substanceand an unlabeled anti-AXL antibody. In this type of assay, thebiological sample, the labeled AXL standard(s) and the anti-AXL antibodyare combined, and the amount of labeled AXL standard bound to theunlabeled anti-AXL antibody is determined. The amount of AXL in thebiological sample is inversely proportional to the amount of labeled AXLstandard bound to the anti-AXL antibody.

Suitable labels for the anti-AXL antibody, secondary antibody and/or AXLstandard used in in vitro diagnostic techniques include, withoutlimitation, various enzymes, prosthetic groups, fluorescent materials,luminescent materials, and radioactive materials. Examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, and acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin;an example of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S, and ³H.

In one aspect, the anti-AXL antibodies of the invention are used in thein vivo imaging of AXL-expressing tissues such as tumors. For in vivomethods, antibody fragments such as, e.g., (Fab′)₂, Fab and Fab′fragments, are particularly advantageous because of their rapiddistribution kinetics.

In vivo imaging can be performed by any suitable technique. For example,an anti-AXL antibody (such as, e.g., a fragment) labeled with ⁹⁹Tc,¹³¹I, ¹¹¹In or other gamma-ray emitting isotope may be used to imageanti-AXL antibody accumulation or distribution in AXL-expressing tissuessuch as tumors with a gamma scintillation camera (e.g., an Elscint Apex409ECT device), typically using low-energy, high resolution collimatoror a low-energy all-purpose collimator. Alternatively, labeling with⁸⁹Zr, ⁷⁶Br, ¹⁸F or other positron-emitting radionuclide may be used toimage anti-AXL antibody or antibody fragment distribution in tumorsusing positron emission tomography (PET). The images obtained by the useof such techniques may be used to assess biodistribution of AXL in apatient, mammal, or tissue, for example in the context of using AXL as abiomarker for the presence of cancer cells. Variations on this techniquemay include the use of magnetic resonance imaging (MRI) to improveimaging over gamma camera techniques. Conventional immunoscintigraphymethods and principles are described in, e.g., [126], [127], and [128].Moreover, such images may also, or alternatively, serve as the basis forsurgical techniques to remove tumors. Furthermore, such in vivo imagingtechniques may allow for the identification and localization of a tumorin a situation where a patient is identified as having a tumor (due tothe presence of other biomarkers, metastases, etc.), but the tumorcannot be identified by traditional analytical techniques. All of thesemethods are features of the present invention.

The in vivo imaging and other diagnostic methods provided by the presentinvention are particularly useful in the detection of micrometastases ina human patient (e.g., a patient not previously diagnosed with cancer ora patient in a period of recovery/remission from a cancer).

In one embodiment, the present invention provides an in vivo imagingmethod wherein an anti-AXL antibody of the present invention isconjugated to a detection-promoting radio-opaque agent, the conjugatedantibody is administered to a host, such as by injection into thebloodstream, and the presence and location of the labeled antibody inthe host is assayed. Through this technique and any other diagnosticmethod provided herein, the present invention provides a method forscreening for the presence of disease-related cells in a human patientor a biological sample taken from a human patient and/or for assessingthe distribution of anti-AXL antibody prior to anti-AXL ADC therapy.

For diagnostic imaging, radioisotopes may be bound to an anti-AXLantibody either directly or indirectly by using an intermediaryfunctional group. Useful intermediary functional groups includechelators, such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid (see for instance [129]).

In addition to radioisotopes and radio-opaque agents, diagnostic methodsmay be performed using anti-AXL antibodies that are conjugated to dyes(such as with the biotin-streptavidin complex), contrast agents,fluorescent compounds or molecules and enhancing agents (e.g.paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g.,[130], which describes MRI techniques and the preparation of antibodiesconjugated to a MRI enhancing agent). Such diagnostic/detection agentsmay be selected from agents for use in MRI, and fluorescent compounds.In order to load an anti-AXL antibody with radioactive metals orparamagnetic ions, it may be necessary to react it with a reagent havinga long tail to which a multiplicity of chelating groups are attached forbinding the ions. Such a tail may be a polymer such as a polylysine,polysaccharide, or another derivatized or derivatizable chain havingpendant groups to which may be bound chelating groups such as, e.g.,porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes,and like groups known to be useful for this purpose. Chelates may becoupled to anti-AXL antibodies using standard chemistries.

Thus, the present invention provides a diagnostic anti-AXL antibody,wherein the anti-AXL antibody is conjugated to a contrast agent (such asfor magnetic resonance imaging, computed tomography, or ultrasoundcontrast-enhancing agent) or a radionuclide that may be, for example, agamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope.

In a further aspect, the invention relates to a kit for detecting thepresence of AXL antigen or a cell expressing AXL, in a sample,comprising:

an anti-AXL antibody, bispecific antibody, or immunoconjugate or ADC ofthe invention; and

instructions for use of the kit.

In one embodiment, the present invention provides a kit for diagnosis ofcancer comprising a container comprising an anti-AXL antibody, and oneor more reagents for detecting binding of the anti-AXL antibody to AXL.Reagents may include, for example, fluorescent tags, enzymatic tags, orother detectable tags. The reagents may also include secondary ortertiary antibodies or reagents for enzymatic reactions, wherein theenzymatic reactions produce a product that may be visualized. In oneembodiment, the present invention provides a diagnostic kit comprisingone or more anti-AXL antibodies, of the present invention in labeled orunlabeled form in suitable container(s), reagents for the incubationsfor an indirect assay, and substrates or derivatizing agents fordetection in such an assay, depending on the nature of the label.Control reagent(s) and instructions for use also may be included.

Diagnostic kits may also be supplied for use with an anti-AXL antibody,such as a conjugated/labeled anti-AXL antibody, for the detection of thepresence of AXL in a tissue sample or host. Thus, the anti-AXL antibodyaccording to the present invention may also be used as, e.g. part of a,companion diagnostic, for example as the primary antibody in animmunohistochemistry assay designed to detect AXL expression in solidtumor, lymph node or other tissue biopsies. Alternatively, the anti-AXLantibodies according to the present invention may be used as the primaryantibody in a flow cytometry-based or immunocytochemistry assay toidentify AXL-expressing cells in blood, bone marrow, fine needlesaspirates, e.g. lymph node aspirates, or peritoneal fluid to identifyAXL-expressing tumor cells. Anti-AXL antibodies according to the presentinvention may be used to identify soluble AXL, e.g. in an ELISA-basedassay. Anti-AXL antibodies according to the present invention may beused as a companion diagnostic, for instance as radioconjugates, thatcan be used for imaging studies in patients. In such diagnostic kits, aswell as in kits for therapeutic uses described elsewhere herein, ananti-AXL antibody typically may be provided in a lyophilized form in acontainer, either alone or in conjunction with additional antibodiesspecific for a target cell or peptide. Typically, a pharmaceuticallyacceptable carrier (e.g., an inert diluent) and/or components thereof,such as a Tris, phosphate, or carbonate buffer, stabilizers,preservatives, biocides, inert proteins, e.g., serum albumin, or thelike, also are included (typically in a separate container for mixing)and additional reagents (also typically in separate container(s)). Incertain kits, a secondary antibody capable of binding to theAXL-specific Ab, which typically is present in a separate container, isalso included. The second antibody is typically conjugated to a labeland formulated in a manner similar to the anti-AXL antibody of thepresent invention. Using the methods described above and elsewhereherein, anti-AXL antibodies may be used to define subsets ofcancer/tumor cells and characterize such cells and related tumortissues.

Anti-Idiotypic Antibodies

In a further aspect, the invention relates to an anti-idiotypic antibodywhich binds to an anti-AXL antibody of the invention as describedherein.

An anti-idiotypic (Id) antibody is an antibody which recognizes uniquedeterminants generally associated with the antigen-binding site of anantibody. An anti-Id antibody may be prepared by immunizing an animal ofthe same species and genetic type as the source of an anti-AXLmonoclonal antibody with the monoclonal antibody against which ananti-Id is being prepared. The immunized animal typically can recognizeand respond to the idiotypic determinants of the immunizing antibody byproducing an antibody to these idiotypic determinants (the anti-Idantibody). Such antibodies are described in for instance U.S. Pat. No.4,699,880. Such antibodies are further features of the presentinvention.

An anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. An anti-anti-Id antibody may be epitopicallyidentical to the original monoclonal antibody, which induced the anti-Idantibody. Thus, by using antibodies to the idiotypic determinants of amonoclonal antibody, it is possible to identify other clones expressingantibodies of identical specificity. Anti-Id antibodies may be varied(thereby producing anti-Id antibody variants) and/or derivatized by anysuitable technique, such as those described elsewhere herein withrespect to AXL-specific antibodies of the present invention. Forexample, a monoclonal anti-Id antibody may be coupled to a carrier suchas keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice.Sera from these mice typically will contain anti-anti-Id antibodies thathave the binding properties similar, if not identical, to anoriginal/parent anti-AXL antibody.

Sequences

TABLE 1 SEQ ID NO: Name Amino acid sequence Comments SEQ ID NO: 1 107 VHEVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYA MNWV HCo12-BalbC RQAPGKGLEWVSTTSGSGAST YYADSVKGRFTISRDNS Ig1 domain KNTLYLQMNSLRAEDTAVYYC AKIWIAFDIWGQGTMV binding Ab TVSS SEQ ID NO: 2 107 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSY LAWYQQ KPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPYT FGQGTKLEIK SEQ ID NO: 3 140 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYA MTWVR QAPGKGLEWVSA ISISGAST FYADSVKGRFTISRDNSKNTLSLQMNSLRAEDTAVYFC RGYSGYVYDAFDI WGQGT MVTVSS SEQ ID NO: 4 140 VLDIQMTQSPSSLSASVGDRVTITCRAS QGISNW LAWYQ QKPEKAPKSLIY AASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYC QQYNSYPLT FGGGTKVEIK SEQ ID NO: 5148 VH EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYA MTWVR HCo12-BalbC QAPGKGLEWVSAISISGGST FYADSVKGRFTISRDNSKN Ig2 domain TLYLQMNSLRAEDTAVYYCRGYSGYVYDAFDF WGQGT binding Ab MVTVSS SEQ ID NO: 6 148 VLDIQMTQSPSSLSASVGDRVTITCRAS QGISNW LAWYQ QKPEKAPKSLIY AASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYC QQYNSYPLT FGGGTKVEIK SEQ ID NO: 7154 VH EVQLLDSGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVR HCo12-BalbC QAPGKGLEWVSAISIGGGNA YYADSVKGRFTISRDNSK FN1 domain NTLYLQMNSLRAADTAVYYCAKPGFIMVRGPLDY WG binding Ab QGALVTVSS SEQ ID NO: 8 154-M103L VHEVQLLDSGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVR QAPGKGLEWVSA ISIGGGNAYYADSVKGRFTISRDNSK NTLYLQMNSLRAADTAVYYC AKPGFILVRGPLDY WGQ GALVTVSS SEQID NO: 9 154 VL EIVLTQSPGTLSLSPGERATLSCRAS QSVSNSY LAWYQQ KPGQAPRLLIYGAS SRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYC QQYGSSPYT FGQGTKLEIK SEQ IDNO: 10 171 VH EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVR HCo17-BalbCQAPGKGLEWVSD ISVSGGST YYADSVKGRFTISRDNSK Ig2 domain NTLYLQMNSLRAEDTAVYYCAKEGYIWFGESLSYAFDI binding Ab WGQGTMVTVSS SEQ ID NO: 11 171 VLEIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQ KPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYC QQYGRSFT FGPGTKVDIK SEQ ID NO: 12172 VH EVQLLESGGGLVQPGGSLRLSCAAS GFTFSNYA MSWV RQAPGKGLEWVSD ISVSGGSTYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYC AKEGYIWFGESLSYAFDI WGQGTMVTVSSSEQ ID NO: 13 172 VL EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYC QQYGRSFTFGPGTKVDIK SEQ ID NO: 14 181 VH EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSD ISVSGGST YYADSVKGRFTISRDNSK NTLYLHMNSLRAEDTAVYYCAKEGYIWFGESLSYAFDI WGQGTMVTVSS SEQ ID NO: 15 181 VHEIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQ KPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYC QQYGRSFT FGPGTKVDIK SEQ ID NO: 16183 VH QVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYY WSWI HCo17-BalbC RQPPGKGLEWIGEINQSGST NYNPSLKSRVTISVDTSKN FN1 domain QFSLKLSSVTAADTSVYYC ASGNWDHFFDYWGQGTLV binding Ab TVSS SEQ ID NO: 17 183-N52Q VHQVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYY WSWI RQPPGKGLEWIGE IQQSGSTNYNPSLKSRVTISVDTSKN QFSLKLSSVTAADTSVYYC ASGNWDHFFDY WGQGTLV TVSS SEQ IDNO: 18 183 VL DIQMTQSPSSVSASVGDRVTITCRAS QGISSW LAWYQ HKPGKAPKLLIY ATSSLQSGVTSRFSGSGSGTDFTLTISSL QPEDFATYYC QQAKSFPWT FGQGTKVEIK SEQ ID NO: 19187 VH QVPLQQWGAGLLKPSETLSLTCAVY GGSFSGYH WSWI RQPPGKGLEWIGE ISHSGRTNYNPSLKSRVTISIDTSKNQ FSLKLSSVTAADTAVYYC ASFITMIRGTIITHFDY WGQGT LVTVSSSEQ ID NO: 20 187 VL DIQMTQSPSSLSASVGDRVTITCRAS QGISSW LAWYQQKPEKAPKSLIY AAS SLQSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYC QQYHSYPYTFGQGTKLEIK SEQ ID NO: 21 608-01 VH QVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYAISWVR QAPGQGLEWMGR IIPIFGIA NYVQKFQGRVTITADKST STAYMELSSLRAEDTAVYYCARRGDYYGSGSPDVFDI W GQGTMVTVSS SEQ ID NO: 22 608-01 VLEIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQ KPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYC QQYGSSYT FGQGTKLEIK SEQ ID NO: 23610-01 VH QVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVR QAPGQGLEWMGR IIPIFGIANYVQKFQGRVTITADKST STAYMELSSLRAEDTAVYYC ARRGNYYGSGSPDVFDI W GQGTMVTVSSSEQ ID NO: 24 610-01 VL EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYC QQYGSSYTFGQGTKLEIK SEQ ID NO: 25 613 VH QVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA INWMHCo20 RQAPGQGLEWMGR IIPIFGIV NYAQKFQGRVTLTADKS Ig1 domainTSTAYMELSSLRSEDTAVYYC ARRGNYYGSGSPDVFDI binding Ab WGQGTMVTVSS SEQ IDNO: 26 613 VL EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQ KPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYC QQYGSSYT FGQGTKLEIK SEQ ID NO: 27613-08 VH QVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA INWM RQAPGQGLEWMGR IIPIFGIVNYAQKFQGRVTLTADKS TSTAYMELSSLRSEDTAVYYC ARRGNYYGSGSPDVFDI WGQGTMVTVSSSEQ ID NO: 28 613-08 VL EIVLTQSPATLSLSPGERATLSCRAS QSVSSY LAWYQQKPGQAPRLLIY DAS NRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYC QQRSNWLT FGGGTKVEIKSEQ ID NO: 29 620-06 VH QVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPIFGIA NYAQKFQGRVTITADKST STAYMELSSLRSEDTAVYYCARRGNYYGSGSPDVFDI W GQGTMVTVSS SEQ ID NO: 30 620-06 VLEIVLTQSPGTLSLSPGERATLSCRAS QSVSSSY LAWYQQ KPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYC QQYGSSYT FGQGTKLEIK SEQ ID NO: 31726 VH QVQLQQWGAGLLKPSETLSLTCAIDG GSFSGYY WSWIR HCo17-BalbC QPPGKGLEWIGEISHSGRT NYNPSLKSRVTISIDTSKNQF FN2 domain SLKLSSVAAADTAVYYCARFITMIRGAIITHFDY WGQGA binding Ab LVTVSS SEQ ID NO: 32 726-M101L VHQVQLQQWGAGLLKPSETLSLTCAID GGSFSGYY WSWIR QPPGKGLEWIGE ISHSGRTNYNPSLKSRVTISIDTSKNQF SLKLSSVAAADTAVYYC ARFITLIRGAIITHFDY WGQGAL VTVSSSEQ ID NO: 33 726 VL DIQMTQSPSSLSASVGDRVTITCRAS QGISSW LAWYQQKPEKAPKSLIY AAS SLQSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYC QQYHSYPYTFGQGTKLEIK SEQ ID NO: 34 733 VH QVQLVESGGGVVQPGRSLRLSCAAS GFSFSTYA MHWVHCo17-BalbC RQAPGKGLEWVAV ISYDGDNK YSADSVKGRFTISRDNS FN1 domainKNTLYLQMNSLRAEDTAVYYC ARGRKLGIDAFDI WGQ binding Ab GTMVTVSS SEQ ID NO:35 733 VL AIQLTQSPSSLSASVGDRVTITCRAS QGISSA LAWYQQK PGKAPKLLIY DASSLESGVPSRFSGSGSGTDFTLTISGLQP EDFATYYC QQFNSYPFT FGPGTKVDIK SEQ ID NO: 36107 VH CDR1 GFTFSSYA SEQ ID NO: 37 107 VH CDR2 TSGSGAST SEQ ID NO: 38107 VH CDR3 AKIWIAFDI SEQ ID NO: 39 107 VL CDR1 QSVSSSY 107 VL CDR2 GASSEQ ID NO: 40 107 VL CDR3 QQYGSSPYT SEQ ID NO: 41 140 VH CDR1 GFTFSSYASEQ ID NO: 42 140 VH CDR2 ISISGAST SEQ ID NO: 43 140 VH CDR3RGYSGYVYDAFDI SEQ ID NO: 44 140 VL CDR1 QGISNW 140 VL CDR2 AAS SEQ IDNO: 45 140 VL CDR3 QQYNSYPLT SEQ ID NO: 46 148 VH CDR1 GFTFSSYA SEQ IDNO: 47 148 VH CDR2 ISISGGST SEQ ID NO: 48 148 VH CDR3 RGYSGYVYDAFDF SEQID NO: 49 148 VL CDR1 QGISNW 148 VL CDR2 AAS SEQ ID NO: 50 148 VL CDR3QQYNSYPLT SEQ ID NO: 51 154 VH CDR1 GFTFSSYA SEQ ID NO: 52 154 VH CDR2ISIGGGNA SEQ ID NO: 53 154 VH CDR3 AKPGFIMVRGPLDY SEQ ID NO: 54154-M103L VH AKPGFILVRGPLDY CDR3 SEQ ID NO: 55 154 VL CDR1 QSVSNSY 154VL CDR2 GAS SEQ ID NO: 56 154 VL CDR3 QQYGSSPYT SEQ ID NO: 57 171 VHCDR1 GFTFSSYA SEQ ID NO: 58 171 VH CDR2 ISVSGGST SEQ ID NO: 59 171 VHCDR3 AKEGYIWFGESLSYAFDI SEQ ID NO: 60 171 VL CDR1 QSVSSSY 171 VL CDR2GAS SEQ ID NO: 61 171 VL CDR3 QQYGRSFT SEQ ID NO: 62 172 VH CDR1GFTFSNYA SEQ ID NO: 63 172 VH CDR2 ISVSGGST SEQ ID NO: 64 172 VH CDR3AKEGYIWFGESLSYAFDI SEQ ID NO: 65 172 VL CDR1 QSVSSSY 172 VL CDR2 GAS SEQID NO: 66 172 VL CDR3 QQYGRSFT SEQ ID NO: 67 181 VH CDR1 GFTFSSYA SEQ IDNO: 68 181 VH CDR2 ISVSGGST SEQ ID NO: 69 181 VH CDR3 AKEGYIWFGESLSYAFDISEQ ID NO: 70 181 VL CDR1 QSVSSSY 181 VL CDR2 GAS SEQ ID NO: 71 181 VLCDR3 QQYGRSFT SEQ ID NO: 72 183 VH CDR1 GGSFSGYY SEQ ID NO: 73 183 VHCDR2 INQSGST SEQ ID NO: 74 183-N52Q VH CDR2 IQQSGST SEQ ID NO: 75 183 VHCDR3 ASGNWDHFFDY SEQ ID NO: 76 183 VL CDR1 QGISSW 183 VL CDR2 ATS SEQ IDNO: 77 183 VL CDR3 QQAKSFPWT SEQ ID NO: 78 187 VH CDR1 GGSFSGYH SEQ IDNO: 79 187 VH CDR2 ISHSGRT SEQ ID NO: 80 187 VH CDR3 ASFITMIRGTIITHFDYSEQ ID NO: 81 187 VL CDR1 QGISSW 187 VL CDR2 AAS SEQ ID NO: 82 187 VLCDR3 QQYHSYPYT SEQ ID NO: 83 608-01 VH CDR1 GGTFSSYA SEQ ID NO: 84608-01 VH CDR2 IIPIFGIA SEQ ID NO: 85 608-01 VH CDR3 ARRGDYYGSGSPDVFDISEQ ID NO: 86 608-01 VL CDR1 QSVSSSY 608-01 VL CDR2 GAS SEQ ID NO: 87608-01 VL CDR3 QQYGSSYT SEQ ID NO: 88 610-01 VH CDR1 GGTFSSYA SEQ ID NO:89 610-01 VH CDR2 IIPIFGIA SEQ ID NO: 90 610-01 VH CDR3ARRGNYYGSGSPDVFDI SEQ ID NO: 91 610-01 VL CDR1 QSVSSSY 610-01 VL CDR2GAS SEQ ID NO: 92 610-01 VL CDR3 QQYGSSYT SEQ ID NO: 93 613 VH CDR1GGTFSSYA SEQ ID NO: 94 613 VH CDR2 IIPIFGIV SEQ ID NO: 95 613 VH CDR3ARRGNYYGSGSPDVFDI SEQ ID NO: 96 613 VL CDR1 QSVSSSY 613 VL CDR2 GAS SEQID NO: 97 613 VL CDR3 QQYGSSYT SEQ ID NO: 98 613-08 VH CDR1 GGTFSSYA SEQID NO: 99 613-08 VH CDR2 IIPIFGIV SEQ ID NO: 100 613-08 VH CDR3ARRGNYYGSGSPDVFDI SEQ ID NO: 101 613-08 VL CDR1 QSVSSY 613-08 VL CDR2DAS SEQ ID NO: 102 613-08 VL CDR3 QQRSNWLT SEQ ID NO: 103 620-06 VH CDR1GGTFSSYA SEQ ID NO: 104 620-06 VH CDR2 IIPIFGIA SEQ ID NO: 105 620-06 VHCDR3 ARRGNYYGSGSPDVFDI SEQ ID NO: 106 620-06 VL CDR1 QSVSSSY 620-06 VLCDR2 GAS SEQ ID NO: 107 620-06 VL CDR3 QQYGSSYT SEQ ID NO: 108 726 VHCDR1 GGSFSGYY SEQ ID NO: 109 726 VH CDR2 ISHSGRT SEQ ID NO: 110 726 VHCDR3 ARFITMIRGAIITHFDY SEQ ID NO: 111 726-M101L VH ARFITLIRGAIITHFDYCDR3 SEQ ID NO: 112 726 VL CDR1 QGISSW 726 VL CDR2 AAS SEQ ID NO: 113726 VL CDR3 QQYHSYPYT SEQ ID NO: 114 733 VH CDR1 GFSFSTYA SEQ ID NO: 115733 VH CDR2 ISYDGDNK SEQ ID NO: 116 733 VH CDR3 ARGRKLGIDAFDI SEQ ID NO:117 733 VL CDR1 QGISSA 733 VL CDR2 DAS SEQ ID NO: 118 733 VL CDR3QQFNSYPFT SEQ ID NO: 119 Ig2 domain VH ISISGXST - wherein X is A or GCDR2 SEQ ID NO: 120 Ig2 domain VH RGYSGYVYDAFDX - wherein X is I or FCDR3 SEQ ID NO: 121 FN2 domain VH GGSFSGYX - wherein X is H or Y CDR1SEQ ID NO: 122 FN2 domain VH AX1FITMIRGX2IITHFDY - wherein X1 is S or R;and CDR3 X2 is T or A SEQ ID NO: 123 FN1 domain VH GFTFSXYA - wherein Xis S or N CDR1 SEQ ID NO: 124 FN1 domain VH ISVSGGST CDR2 SEQ ID NO: 125FN1 domain VH AKEGYIWFGESLSYAFDI CDR3 SEQ ID NO: 126 Ig1 domain VHIIPIFGIX - wherein X is A or V CDR2 SEQ ID NO: 127 Ig1 domain VHARRGXYYGSGSPDVFDI - wherein X is D or N CDR3 SEQ ID NO: 128 Ig1 domainVL QSVXSSY - wherein X is S or del CDR1 Ig1 domain VL XAS - wherein X isD or G CDR2 SEQ ID NO: 129 Ig1 domain VL QQX1X2X3X4X5T - wherein X1 is Ror Y; X2 is S or CDR3 G; X3 is N or S; X4 is W or S; and X5 is L or YSEQ ID NO: 130 Human AXL protein MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEES(Swissprot P30530) PFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTP SPAQPADRGSPAAPGQEDGA SEQ ID NO:131 Mus musculus AXL MAWRCPRMGRVPLAWCLALCGWACMYPYDVPDYAAHKDTQTEAGSPFVGNPGNITGARGLTGTLRCELQVQGEPPEVVWLRDGQILELADNTQTQVPLGEDWQDEWKVVSQLRISALQLSDAGEYQCMVHLEGRTFVSQPGFVGLEGLPYFLEEPEDKAVPANTPFNLSCQAQGPPEPVTLLWLQDAVPLAPVTGHSSQHSLQTPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQRPHHLHVVSRQPTELEVAWTPGLSGIYPLTHCNLQAVLSDDGVGIWLGKSDPPEDPLTLQVSVPPHQLRLEKLLPHTPYHIRISCSSSQGPSPWTHWLPVETTEGVPLGPPENVSAMRNGSQVLVRWQEPRVPLQGTLLGYRLAYRGQDTPEVLMDIGLTREVTLELRGDRPVANLTVSVTAYTSAGDGPWSLPVPLEPWRPGQGQPLHHLVSEPPPRAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAG RYVLCPSTTPSPAQPADRGSPAAPGQEDGA SEQID NO: 132 Homo sapiens AXL - MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEES Musmusculus Ig1 PFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRD domainGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDKAVPANTPFNLSCQAQGPPEPVTLLWLQDAVPLAPVTGHSSQHSLQTPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDR HKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRY VLCPSTTPSPAQPADRGSPAAPGQEDGA SEQID NO: 133 Homo sapiens AXL - MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEES Musmusculus Ig2 PFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRD domainGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDKAVPANTPFNLSCQAQGPPEPVTLLWLQDAVPLAPVTGHSSQHSLQTPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDR HKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRY VLCPSTTPSPAQPADRGSPAAPGQEDGA SEQID NO: 134 Homo sapiens AXL - MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEES Musmusculus PFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRD FN1 domainGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQRPHHLHVVSRQPTELEVAWTPGLSGIYPLTHCNLQAVLSDDGVGIWLGKSDPPEDPLTLQVSVPPHQLRLEKLLPHTPYHIRISCSSSQGPSPWTHWLPVETTEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDR HKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRY VLCPSTTPSPAQPADRGSPAAPGQEDGA SEQID NO: 135 Homo sapiens AXL - MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEES Musmusculus PFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRD FN2 domainGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENVSAMRNGSQVLVRWQEPRVPLQGTLLGYRLAYRGQDTPEVLMDIGLTREVTLELRGDRPVANLTVSVTAYTSAGDGPWSLPVPLEPWRPGQGQPLHHLVSEPPPRAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTP SPAQPADRGSPAAPGQEDGA SEQ ID NO:136 511 VH EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYA MNWV Ig2 domainRQAPGKGLEWVSG ISGSGGHT YHADSVKGRFTISRDNS binding AbKNTLYLQMNSLRAEDTAVYYC AKDRYDILTGYYNLLDY WGQGTLVTVSS SEQ ID NO: 137 511VH CDR1 GFTFSSYA SEQ ID NO: 138 511 VH CDR2 ISGSGGHT SEQ ID NO: 139 511VH CDR3 AKDRYDILTGYYNLLDY SEQ ID NO: 140 511 VLDIQMTQSPSSLSASVGDRVTITCRAS QGISSW LAWYQQ KPEEAPKSLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYC QQYNSYPLT FGGGAKVEIK SEQ ID NO:141 511 VL CDR1 QGISSW 511 VL CDR2 AAS SEQ ID NO: 142 511 VL CDR3QQYNSYPLT SEQ ID NO: 143 061 VH QVQLVQSGAEVKKPGASVKVSCKASGYAFTGYGISWVRIg1 domain QAPGQGLEWIGWISAYNGNTNYVQNLQDRVTMTTDT binding AbSTSTAYMELRSLRSDDTAVYYCARDHISMLRGIIIRNYW GQGTLVTVSS SEQ ID NO: 144 061 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPE DFAVYYCQQRSSWPRLTFGGGTKVEIKSEQ ID NO: 145 137 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSRYAISWVRQAPGQGLEWMGRIIPIVGIANYAQKFQGRVTLTADKSTSTAYMELSSLRSEDTAVYYCAREAGYSSSWYAEYFQHW GQGTLVTVSS SEQ ID NO: 146 137 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIYGASSRATGFPDRFSGSGSGTDFTLTISRL EPEDFAVYYCQQYGSSPYTFGQGTKLEIKSEQ ID NO: 147 Cynomolgus AWRCPRMGRVPLAWCLALCGWVCMAPRGTQAEESP monkey AXLFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDG (GenBankQILELADSTQTQVPLGEDEQDDWIVVSQLRIASLQLSDA numberGQYQCLVFLGHQNFVSQPGYVGLEGLPYFLEEPEDRTV HB387229.1)AANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRNLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTLQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKETSAPAFSWPWWYILLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQLDPKDSCSCLTSAEVHPAGRYVLCPSTAPS PAQPADRGSPAAPGQEDGA

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

EXAMPLES Example 1—Immunization and Generation of AXL Antibodies

Expression Constructs for AXL

The following codon-optimized constructs for expression of variousfull-length AXL variants were generated: human (Homo sapiens) AXL(Genbank accession no. NP_068713.2), human-cynomolgus monkey chimericAXL in which the human extracellular domain (ECD) was replaced with theECD of cynomolgus monkey (Macaca fascicularis) AXL (translation ofGenbank accession HB387229.1; aa 1-447), human-mouse chimeric AXL inwhich the human ECD was replaced with the ECD of mouse (Mus musculus)AXL (Genbank accession NP_033491.2; aa 1-441), human-mouse chimeric AXLin which the human Ig-like domain I (aa 1-134, also termed “Ig1 domain”herein) was replaced with the Ig-like domain I of mouse AXL, human-mousechimeric AXL in which the human Ig-like domain II (aa 148-194, alsotermed “Ig2 domain” herein) was replaced by the Ig-like domain II ofmouse AXL, human-mouse chimeric ALX in which the human FNIII-like domainI (aa 227-329, also termed “FN1 domain” herein) was replaced with theFNIII-like domain I of mouse AXL, human-mouse chimeric AXL in which thehuman FNIII-like domain II (aa 340-444, also termed “FN2 domain” herein)was replaced by the FNIII-like domain II of mouse AXL. In addition, thefollowing codon-optimized constructs for various AXL ECD variants weregenerated: the extracellular domain (ECD) of human AXL (aa 1-447) with aC-terminal His tag (AXLECDHis), the FNIII-like domain II of human AXL(aa 327-447) with a N-terminal signal peptide and a C-terminal His tag(AXL-FN2ECDHis), and the Ig1- and Ig2-like domains of human AXL (aa1-227) with a C-terminal His tag (AXL-Ig12ECDHis).

The constructs contained suitable restriction sites for cloning and anoptimal Kozak (GCCGCCACC) sequence [141]. The constructs were cloned inthe mammalian expression vector pcDNA3.3 (Invitrogen).

AXL Expression in EL4 Cells

EL4 cells were stable transfected with the pcDNA3.3 vector containingthe full human AXL coding sequence and stable clones were selected afterselection with the antibiotic agent, G418, (Geneticin).

Purification of His-Tagged AXL

AXLECDHis, AXL-FN2ECDHis, and AXL-Ig12ECDHis were expressed in HEK-293Fcells. The His-tag enables purification with immobilized metal affinitychromatography. In this process, a chelator fixed onto thechromatographic resin is charged with Co²⁺ cations. His-tagged proteincontaining supernatants were incubated with the resin in batch mode(i.e. solution). The His-tagged protein binds strongly to the resinbeads, while other proteins present in the culture supernatant do notbind or bind weakly compared to the His-tagged proteins. Afterincubation the beads are retrieved from the supernatant and packed intoa column. The column is washed in order to remove weakly bound proteins.The strongly bound His-tagged proteins are then eluted with a buffercontaining imidazole, which competes with the binding of His to Co²⁺.The eluent is removed from the protein by buffer exchange on a desaltingcolumn.

Immunization Antibodies IgG1-AXL-061, IgG1-AXL-107, IgG1-AXL-183,IgG1-AXL-613, and IgG1-AXL-726 were derived from the followingimmunizations: HCo12-BalbC (IgG1-AXL-107), HCo17-BalbC (IgG1-AXL-183,IgG1-AXL-726) and HCo20 (IgG1-AXL-061, IgG1-AXL-613) transgenic mice(Medarex, San Jose, Calif., USA) which were immunized alternatinglyintraperitoneally (IP) with 20 μg of the AXLECDHis protein(IgG1-AXL-511, IgG1-AXL-613, IgG1-AXL-183), 20 μg AXL-FN2ECDHIS plus 20μg AXL-Ig12ECDHis (IgG1-AXL-726), or 20 μg AXL-Ig12ECDHis (IgG1-AXL-107)and subcutaneously (SC; at the tail base) with the same protein, with aninterval of 14 days. In total 8 immunizations were performed: 4 IP and 4SC immunizations. For most immunizations, the first immunization wasperformed in complete Freunds' adjuvant (CFA; Difco Laboratories,Detroit, Mich., USA) and all subsequent immunizations in incompleteFreunds' adjuvant (IFA; Difco Laboratories, Detroit, Mich., USA).Antibody IgG1-AXL-183 was derived from immunizations that were allperformed in Sigma adjuvant system (Sigma-Aldrich, St. Louis, Mo., USA).

Antibodies IgG1-AXL-137, IgG1-AXL-148, IgG1-AXL-154, IgG1-AXL-171, andIgG1-AXL-733 were derived from the following immunizations: HCo12-BalbC(IgG1-AXL-137, IgG1-AXL-148), HCo17-BalbC (IgG1-AXL-154, IgG1-AXL-733),and HCo20-BalbC (IgG1-AXL-171) transgenic mice (Medarex, San Jose,Calif., USA) were immunized with 20 μg of the AXLECDHis protein in CFA.Subsequently, mice were immunized alternating intraperitoneally (IP)with EL4 cells transfected with full length human AXL in PBS andsubcutaneously (SC; at the tail base) with the AXLECDHis protein in IFA,with an interval of 14 days.

Mice with at least two sequential AXL specific antibody titers of 200(serum dilutions of 1/200) or higher, detected in the antigen specificscreening FMAT assay as described below, were boosted 3-4 days prior tofusion (10 μg of AXL-derived protein in PBS injected intravenously).

Homogeneous Antigen Specific Screening Assay

The presence of anti-AXL antibodies in sera of immunized mice or HuMab(human monoclonal antibody) hybridoma or transfectoma culturesupernatant was determined by homogeneous antigen specific screeningassays using Fluorometric Micro volume Assay Technology (FMAT; AppliedBiosystems, Foster City, Calif., USA). For this, two different testdesigns with combinations of either 4 or 8 cell based assays were used.

The 4 cell based assay test design was used for the testing of sera fromimmunized mice and as primary screening test for hybridoma ortransfectoma culture supernatant. In the 4 assay test design sampleswere analyzed for binding of human antibodies to A431 (DSMZ) andMDA-MB-231 cells (both expressing AXL at the cell surface) as well asbinding to TH1021-AXL (HEK-293F cells transiently expressing full lengthhuman AXL; produced as described above) and HEK293 wild-type cells(negative control which does not express AXL), respectively.

Hybridoma or transfectoma culture supernatant samples were additionallysubjected to an 8 cell based assay test design. In the 8 assay testdesign samples were analyzed for binding of human antibodies toTH1021-hAXL (HEK-293F cells transiently expressing the human AXL),TH1021-cAXL (HEK-293F cells transiently expressing human-cynomolgus AXLchimeras in which the human ECD had been replaced with the ECD ofcynomolgus monkey AXL), TH1021-mAXL (HEK-293F cells transientlyexpressing human-mouse AXL chimeras in which the human ECD had beenreplaced with the ECD of mouse AXL), TH1021-mIg1 (HEK-293F cellstransiently expressing the human AXL with the Ig-like domain I beingreplaced by the Ig-like domain I of mouse AXL), TH1021-mIg2 (HEK-293Fcells transiently expressing human AXL with the Ig-like domain II beingreplaced by the Ig-like domain II of mouse AXL), TH1021-mFN1 (HEK-293Fcells transiently expressing human AXL with the FNIII-like domain Ibeing replaced by the FNIII-like domain I of mouse AXL), TH1021-mFN2(HEK-293F cells transiently expressing human AXL with the FNIII-likedomain II being replaced by the FNIII-like domain II of mouse AXL), andHEK293 wild-type cells (negative control which does not express AXL),respectively.

Samples were added to the cells to allow binding to AXL. Subsequently,binding of HuMab was detected using a fluorescent conjugate (Goatanti-Human IgG Fc gamma-DyLight649; Jackson ImmunoResearch). The AXLspecific humanized mouse antibody A0704P (produced in HEK-293F cells)was used as a positive control and HuMab-mouse pooled serum andChromPure Human IgG, whole molecule (Jackson ImmunoResearch),respectively, were used as negative controls. The samples were scannedusing an Applied Biosystems 8200 Cellular Detection System (8200 CDS)and mean fluorescence was used as read-out. Samples were stated positivewhen counts were higher than 50 and counts x fluorescence was at leastthree times higher than the negative control.

HuMab Hybridoma Generation

The HuMab mouse with sufficient antigen-specific titer development(described above) was sacrificed and the spleen and lymph nodes flankingthe abdominal aorta and vena cava were collected. Fusion of splenocytesand lymph node cells to a mouse myeloma cell line (SP2.0 cells) was doneby electrofusion using a CytoPulse CEEF 50 Electrofusion System(Cellectis, Paris, France), essentially according to the manufacturer'sinstructions. Next, the primary wells were sub-cloned using the ClonePixsystem (Genetix, Hampshire, UK). To this end, specific primary wellhybridomas were seeded in semisolid medium made from 40% CloneMedia(Genetix, Hampshire, UK) and 60% HyQ 2× complete media (Hyclone,Waltham, USA). The sub clones were retested according to theantigen-specific binding assay as described above and scanned using theIsoCyte sytem (Molecular Devices, LLC, Sunnyvale, Calif.). IgG levelswere measured using an Octet (Fortebio, Menlo Park, USA) in order toselect the best producing clone per primary well for further expansion.Further expansion and culturing of the resulting HuMab hybridomas weredone based upon standard protocols (e.g. as described in Coligan J. E.,Bierer, B. E., Margulies, D. H., Shevach, E. M. and Strober, W., eds.Current Protocols in Immunology, John Wiley & Sons, Inc., 2006). Clonesderived by this process were designated PC1021.

Mass Spectrometry of Purified Antibodies

Small 0.8 ml aliquots of antibody containing hybridoma supernatant from6-well or Hyperflask stage were purified using PhyTip columns containingProtein G resin (PhyNexus Inc., San Jose, USA) on a Sciclone ALH 3000workstation (Caliper Lifesciences, Hopkinton, USA). The PhyTip columnswere used according to manufacturer's instructions, but buffers werereplaced by: Binding Buffer PBS (B. Braun, Medical B.V., Oss,Netherlands) and Elution Buffer 0.1M Glycine-HCl pH 2.7 (Fluka Riedel-deHaën, Buchs, Germany). After purification, samples were neutralized with2M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands).Alternatively, in some cases larger volumes of culture supernatant werepurified using Protein A affinity column chromatography.

After purification, the samples were placed in a 384-well plate (Waters,100 μl square well plate, part#186002631). Samples were deglycosylatedovernight at 37° C. with N-glycosidase F. DTT (15 mg/ml) was added (1μl/well) and incubated for 1 h at 37° C. Samples (5 or 6 μl) weredesalted on an Acquity UPLC™ (Waters, Milford, USA) with a BEH300 C18,1.7 μm, 2.1×50 mm column at 60° C. MQ water and LC-MS grade acetonitrile(Biosolve, cat no 01204101, Valkenswaard, The Netherlands) with both0.1% formic acid (Fluka, cat no 56302, Buchs, Germany), were used asEluent A and B, respectively. Time-of-flight electrospray ionizationmass spectra were recorded on-line on a micrOTOF™ mass spectrometer(Bruker, Bremen, Germany) operating in the positive ion mode. Prior toanalysis, a 900-3000 m/z scale was calibrated with ES tuning mix(Agilent Technologies, Santa Clara, USA). Mass spectra were deconvolutedwith DataAnalysis™ software v. 3.4 (Bruker) using the Maximal Entropyalgorithm searching for molecular weights between 5 and 80 kDa.

After deconvolution the resulting heavy and light chain masses (underreducing conditions) for all samples were compared in order to findduplicate antibodies. In the comparison of the heavy chains the possiblepresence of C-terminal lysine variants was taken into account. Thisresulted in a list of unique antibodies, where unique is defined as aunique combination of heavy and light chains. In case duplicateantibodies were found, the results from other tests were used to decidewhich antibody was the best material to continue experiments with.

Sequence Analysis of the AXL Antibody Variable Domains and Cloning inExpression Vectors

Total RNA was prepared from 0.2 to 5×10⁶ hybridoma cells and5′-RACE-Complementary DNA (cDNA) was prepared from 100 ng total RNA,using the SMART RACE cDNA Amplification kit (Clontech), according to themanufacturer's instructions. VH and VL coding regions were amplified byPCR and cloned directly, in frame, in the pG1f and pKappa expressionvectors, by ligation independent cloning (Aslanidis, C. and P. J. deJong, Nucleic Acids Res 1990; 18(20): 6069-74). For each antibody, 12 VLclones and 12 VH clones were sequenced. The resulting sequences areshown in the Table 1. CDR sequences were defined according to IMGT [22]and [23]. Clones with a correct Open Reading Frame (ORF) were selectedfor further study and expression. Vectors of all combinations of heavychains and light chains that were found were transiently co-expressed inFreestyle™ 293-F cells using 293fectin.

For antibodies IgG1-AXL-154, IgG1-AXL-183 and IgG1-AXL-726, thefollowing variants with point mutations in the variable domains weregenerated: IgG1-AXL-154-M103L, IgG1-AXL-183-N52Q and IgG1-AXL-726-M101L.Mutants were generated by site-directed mutagenesis using theQuickchange II mutagenesis kit (Stratagene).

AXL Control Antibodies

In some of the Examples a comparison antibody against AXL was used(IgG1-YW327.652) that have been previously described [142] and [143].The VH and VL sequences for these AXL-specific antibodies were clonedinto the pG1f and pKappa expression vectors.

b12 Antibody

In some of the examples the antibody b12, a gp120 specific antibody[144] was used as a negative control.

Expression

Antibodies were expressed as IgG1,κ. Plasmid DNA mixtures encoding bothheavy and light chains of antibodies were transiently transfected toFreestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen,US) essentially as described by the manufacturer.

Purification of Antibodies

Culture supernatant was filtered over 0.2 μm dead-end filters, loaded on5 mL MabSelect SuRe columns (GE Health Care) and eluted with 0.1 Msodium citrate-NaOH, pH 3. The eluate was immediately neutralized with2M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mM NaH2PO4, 140 mMNaCl, pH 7.4 (B.Braun). Alternatively, subsequent to purification, theeluate was loaded on a HiPrep Desalting column and the antibody wasexchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B.Braun) buffer.After dialysis or exchange of buffer, samples were sterile filtered over0.2 μm dead-end filters. Purity was determined by SDS-PAGE and IgGconcentration was measured using an Octet (Fortebio, Menlo Park, USA).Purified antibodies were stored at 4° C.

The antibody IgG1-AXL-511 was generated by the following method:

Expression Constructs for AXL

The following codon-optimized constructs for expression of variousfull-length AXL variants were generated: human (Homo sapiens) AXL(Genbank accession no. NP_068713.2), human-cynomolgus monkey chimericAXL in which the human extracellular domain (ECD) was replaced with theECD of cynomolgus monkey (Macaca fascicularis) AXL (translation ofGenbank accession HB387229.1; aa 1-447), human-mouse chimeric AXL inwhich the human ECD was replaced with the ECD of mouse (Mus musculus)AXL (Genbank accession NP_033491.2; aa 1-441), human-mouse chimeric AXLin which the human Ig-like domain I (aa 1-147, also termed “Ig1 domain”herein) was replaced with the Ig-like domain I of mouse AXL, human-mousechimeric AXL in which the human Ig-like domain II (aa 148-227, alsotermed “Ig2 domain” herein) was replaced by the Ig-like domain II ofmouse AXL, human-mouse chimeric ALX in which the human FNIII-like domainI (aa 228-326, also termed “FN1 domain” herein) was replaced with theFNIII-like domain I of mouse AXL, human-mouse chimeric AXL in which thehuman FNIII-like domain II (aa 327-447, also termed “FN2 domain” herein)was replaced by the FNIII-like domain II of mouse AXL. In addition, thefollowing codon-optimized constructs for various AXL ECD variants weregenerated: the extracellular domain (ECD) of human AXL (aa 1-447) with aC-terminal His tag (AXLECDHis), the FNIII-like domain II of human AXL(aa 327-447) with a N-terminal signal peptide and a C-terminal His tag(AXL-FN2ECDHis), and the Ig1- and Ig2-like domains of human AXL (aa1-227) with a C-terminal His tag (AXL-Ig12ECDHis).

The constructs contained suitable restriction sites for cloning and anoptimal Kozak (GCCGCCACC) sequence (Kozak et al. (1999) Gene 234:187-208). The constructs were cloned in the mammalian expression vectorpcDNA3.3 (Invitrogen).

AXL Expression in EL4 Cells

EL4 cells were stable transfected with the pcDNA3.3 vector containingthe full length human AXL coding sequence and stable clones wereselected after selection with the antibiotic agent, G418, (Geneticin).

Purification of His-Tagged AXL

AXLECDHis, AXL-FN2ECDHis, and AXL-Ig12ECDHis were expressed in HEK293Fcells and purified with immobilized metal affinity chromatography.

Immunization

Material from 4 transgenic mice expressing human antibody gene sequenceswas used for selecting antibodies. Mice immunized with variousimmunization protocols and with various antibody responses and yieldingvarious numbers of antibodies from the traditional hybridoma processwere chosen. Mouse A (3.5% hits in the hybridoma process) was anHCo17-BALB/c transgenic mouse (Bristol-Myers Squibb, Redwood City,Calif., USA) was immunized alternatingly intraperitoneally (IP) with 20μg AXL-FN2ECDHIS plus 20 μg AXL-Ig12ECDHis) and subcutaneously (SC) atthe tail base) with the same protein, with an interval of 14 days. Intotal 8 immunizations were performed: 4 IP and 4 SC immunizations. Formost immunizations, the first immunization was performed in completeFreunds' adjuvant (CFA; Difco Laboratories, Detroit, Mich., USA) and allsubsequent immunizations in incomplete Freunds' adjuvant (IFA; DifcoLaboratories, Detroit, Mich., USA). Mouse B (0% hits in the hybridomaprocess) was a HCo12 transgenic mouse (Medarex) immunized with 20 μg ofthe AXLECDHis protein using a similar immunization protocol as mouse A.Mouse C (38% hits in the hybridoma process) was a HCo12-BALB/c mouseimmunized alternating intraperitoneally (IP) with EL4 cells transfectedwith full length human AXL in PBS and subcutaneously (SC; at the tailbase) with the AXLECDHis protein in IFA, with an interval of 14 days.Mouse D (0% hits in the hybridoma process) was a HCo12 transgenic mouse(Medarex) immunized with 20 μg of the AXL-Ig12ECDHis protein in using asimilar immunization protocol as mouse A.

Mice with at least two sequential AXL specific antibody titers of 200(serum dilutions of 1/200) or higher, were boosted 3-4 days prior tofusion (10 μg of AXL-derived protein in PBS injected intravenously).

Isolation of RNA from Spleen Cells

Total RNA was isolated from spleen cells using the Mini RNA easy kit(Qiagen). First strand cDNA for 5′-RACE was synthesized using 150 ng ofRNA using the SMART RACE cDNA Amplification kit (Clontech, MountainView, Calif., USA), PrimeScript Reverse Transcriptase (Clontech) and theSMART HA oligo and oligodT as primers. VL encoding regions wereamplified by PCR using Advantage 2 polymerase (Clontech), the primersRACEkLIC4shortFW2 (320 nM), RACEkLIC4LongFW2 (80 nM) andRACEkLICRV_PmIA3 (400 nM), performing 35 cycles of 30 seconds at 95° C.,and 1 minute at 68° C. VH encoding regions were amplified by PCR usingPfu Ultra II Fusion HS DNA polymerase (Stratagene), the primersRACEG1LIC3shortFW (320 nM), RACEG1LIC3longFW (80 nM) and RACEG1LIC3RV2(400 nM), performing 40 cycles of 20 seconds at 95° C., 20 seconds at66° C. and 30 seconds at 72° C., ending with a finale extension step of3 minutes at 72° C. VH or VL encoding PCR products were separated usingagarose gel electrophoresis and DNA products of the expected size werecut from the gel and purified using the Qiagen MiniElute kit. VH and VLcoding regions amplified by PCR were cloned, in frame, in the mammalianexpression vectors pG1f (containing the human IgG1 constant regionencoding DNA sequence) for the VH region and pKappa (containing thekappa light chain constant region encoding DNA sequence) for the VLregion, by ligation independent cloning (Aslanidis, C. and P. J. deJong, Nucleic Acids Res 1990; 18(20): 6069-74) in E. coli strain DH5αT1R(Life technologies), yielding single bacterial colonies each containinga single HC or LC expression vector.

Primer Sequences

Primer name Primer sequence SMARTIIA 5′-AAGCAGTGGTATCAACGCAGAGTACGCGGGRACEkLIC4shortFW2 5′-ACGGACGGCAGGACCACT RACEkLIC4LongFW25′-ACGGACGGCAGGACCACTAAGCAGGGTATCAACGCAGA RACEkLICRV_PmIA35′-CAGCAGGCACACCACTGAGGCAGTTCCAGATTTC RACEG1LIC3shortFW5′-ACGGACGGCAGGACCAGT RACEG1LIC3longFW5′-ACGGACGGCAGGACCAGTAAGCAGTGGTATCAACGCAGAGT RACEG1LIC3RV2 5-GGAGGAGGGCGCCAGTGGGAAGACCGA CMV P f (RRA2) 5′-GCCAGATATACGCGTTGACA TKpA r (RRA2) 5′-GATCTGCTATGGCAGGGCCTLEE PCR

Linear expression elements (LEE's) were produced by amplifying thefragment containing the CMV promoter, HC or LC encoding regions and thepoly A signal containing elements from the expression plasmids. For thisthe regions were amplified using Accuprime Taq DNA polymerase (LifeTechnologies) and the primers CMVPf(Bsal)2 and TkpA(Bsal)r, performing35 cycles of 45 seconds at 94° C., 30 seconds at 55° C. and 2 (LC) or 3(HC) minutes at 68° C., using material of E. coli (strain DH5α)colonies, containing the plasmids, as a DNA template.

Transient Expression in HEK-293 Cells

Antibodies were expressed as IgG1,κ. Plasmid DNA mixtures encoding bothheavy and light chains of antibodies were transiently transfected inFreestyle 293-F (HEK293F) cells (Life technologies, USA) using 293fectin(Life technologies) essentially as described by Vink, T., et al. (2014)(‘A simple, robust and highly efficient transient expression system forproducing antibodies’, Methods, 65 (1), 5-10).

For LEE expression of Abs 1 μl of the HC LEE PCR reaction mixture, 1 μlof the LC PCR reaction mixture and 1 μl of a 30 ng/μl enhancing mixcontaining a mix of 3 expression enhancing plasmids as described inVink, T., et al. (2014), were mixed and transfected in HEK293F cells ina total volume of 100 μl using 293 fectin as transfection reagent,according to the instructions of the manufacturer (Life technologies),using 96 well plates as vessel, essentially as described supra.

AXLECDHis ELISA

ELISA plates (Greiner, Netherlands) were coated with 100 μl/well of 0.5μg/ml AXLECDHis in Phosphate buffered saline (PBS) and incubated for 16hours at room temperature (RT). The coating solution was removed and thewells were blocked by adding 150 μl PBSTC (PBS containing 0.1% tween-20and 2% chicken serum) well and incubating for 1 hour at RT. The plateswere washed three times with 300 μl PBST (PBS containing 0.1%tween-20)/well and 100 μl of test solution was added, followed by anincubation of 1 hour at RT. After washing three times with 300 μl ofPBST/well, 100 μl antibody goat anti human IgG coupled with horse radishperoxidase (diluted 1/3000) was added and incubated for 1 hour at RT.After washing three times with 300 μl of PBST/well, 100 μl of ABTS (1mg/ml) solution was added and incubated at RT until sufficient signalwas observed and the reaction was stopped by adding 100 μl of 2% oxalicacid solution. 96 well plates were measured on an ELISA reader at 405nm.

Diversity Screen

Samples were analyzed for binding of antibodies to TH1021-hAXL (HEK293Fcells transiently expressing the human AXL), TH1021-cAXL (HEK293F cellstransiently expressing human-cynomolgus AXL chimeras in which the humanECD had been replaced with the ECD of cynomolgus monkey AXL),TH1021-mAXL (HEK293F cells transiently expressing human-mouse AXLchimeras in which the human ECD had been replaced with the ECD of mouseAXL), TH1021-mIg1 (HEK293F cells transiently expressing the human AXLwith the Ig-like domain I being replaced by the Ig-like domain I ofmouse AXL), TH1021-mIg2 (HEK293F cells transiently expressing human AXLwith the Ig-like domain II being replaced by the Ig-like domain II ofmouse AXL), TH1021-mFN1 (HEK293F cells transiently expressing human AXLwith the FNIII-like domain I being replaced by the FNIII-like domain Iof mouse AXL), TH1021-mFN2 (HEK293F cells transiently expressing humanAXL with the FNIII-like domain II being replaced by the FNIII-likedomain II of mouse AXL), and HEK293F cells (negative control which doesnot express AXL), respectively.

Samples from the LEE expression were added to the cells to allow bindingto the various AXL constructs. Subsequently, binding of antibodies wasdetected using a fluorescent conjugate (Goat anti-Human IgG Fcgamma-DyLight649; Jackson ImmunoResearch). The samples were scannedusing an Applied Biosystems 8200 Cellular Detection System (8200 CDS)and mean fluorescence was used as read-out. Samples were stated positivewhen counts were higher than 50 and counts x fluorescence was at leastthree times higher than the negative control.

Provision of HC and LC Pools:

For each mouse, 352 HC expression vector containing bacterial coloniesand 384 LC expression vector containing bacterial colonies were pickedand amplified by LEE PCR. Part of the LEE reaction was sequenced(AGOWA). The percentage proper VH insert containing constructs differedlargely between the 4 mice, mouse A (50%), mouse B (23%), mouse C (90%)and mouse D (14%) and resembled the variation of hits obtained in thehybridoma process, see supra. The HC diversity in the mice with only alimited amount of proper inserts were dominated by a large group ofidentical HCs, 65/83 in mouse B and 46/49 in mouse D. For mouse B and Dthe unique HCs (9 for mouse B, 4 for mouse D) were selected. For mouse Aand C no selection was made.

Co-Transfection of HCs with a LC Pool

The single HC encoding LEE's were co-transfected with a pool of 96 LCencoding LEE's using the LEE transfection protocol.

HC Selection of AXL Binding Antibodies

For mouse B and D, supernatants from the LEE co-transfections of thesingle HC with the pooled LCs were analyzed for AXL binding of theproduced antibody mixtures by the AXL ELISA. 7 of the 9 HCs from mouse Bresulted in AXL binding and 4 out of 4 of the HC from mouse D resultedin AXL binding.

For mouse A and C supernatants from the LEE co-transfections of thesingle HC with the pooled LCs were analyzed for AXL binding of theproduced antibody mixtures by the diversity screen. This screen enabledboth the identification of AXL binding HCs and a rough epitope mapping,by identifying the loss of binding of antibodies to AXL variants. Frommouse A approximately 40% of the HCs bound to human AXL, most of whichlost binding either to the Ig1 or FNIII-2 domain, when these domainswere replaced by the mouse equivalent. From mouse C approximately 70% ofthe HCs bound to human AXL, most of which lost binding either to the Ig1or Ig2 domain, when these domains were replaced by the mouse equivalent.Based on binding as determined by AXL ELISA or the diversity screen, HCsequence information and loss of binding to specific AXL domains in thediversity screen a total of 12 unique HCs were selected fordetermination of the best LC.

Co-Transfection of HCs with Single LCs

Each single HC LEE of the 12 unique selected HCs was co-transfected with96 single LC LEEs from the LC pool of the corresponding mice.

LC Selection of AXL Binding Antibodies

Supernatants of the LEE expression of the single HC/LC combinations wereanalyzed for AXL binding of the produced antibody by the AXL ELISA. Foreach HC at least 6 LCs were found and a single LC was selected as best,based on both the ELISA results and the LC sequence information. AXLbinding antibodies were identified from all 4 mice, even the mice whichwere not successful in the hybridoma process.

Binding Affinity of Antibody 511

The affinity of one anti-AXL antibody (clone 511) was determined.

Affinity was determined using Bio-Layer Interferometry on a ForteBioOctetRED384. Anti-human Fc Capture (AHC) biosensors (ForteBio,Portsmouth, UK; cat no. 18-5064) were loaded for 150 s with hIgG (1μg/mL) aiming at a loading response of 1 nm. After a baseline (150 s)the association (1000 s) and dissociation (2000 s) of AXLECDHis (asdescribed in Example 1) was determined, using a concentration range of10 μg/mL-0.16 μg/mL (218 nM-3 nM) with 2-fold dilution steps. Forcalculations, the theoretical molecular mass of AXLECDHis based on theamino acid sequence was used, i.e. 46 kDa. Experiments were carried outon an OctetRED384, while shaking at 1000 rpm and at 30° C. Each antibodywas tested in three independent experiments.

Data was analyzed with ForteBio Data Analysis Software v7.0.3.1, usingthe 1:1 model and a global full fit with 1000 s association time and1000 s dissociation time unless stated otherwise. A dissociation time of1000 s (instead of the 2000 s dissociation time that was acquired) wasused since this resulted in better fits. Data traces were corrected bysubtraction of a reference curve (antibody without AXLECDHis), theY-axis was aligned to the last 5 s of the baseline, and interstepcorrection as well as Savitzky-Golay filtering was applied.

The affinity (K_(D)) of clone 511 for AXL was 23*10⁻⁹M (k_(on) 1.7*10⁵1/Ms and a k_(dls) of 3.9*10⁻³ 1/s).

Duostatin-3 Synthesis.

Preparation of Compound 3:

To a solution of Boc-L-phenylalanine 1 (5.36 g, 20.2 mmol) in 30 mL ofmethylene chloride (DCM), carbonyldiimidazole (CDI, 4.26 g, 26.3 mmol)was added and stirred for 1 hour. Then added a solution of 2 (3.67 g,30.3 mmol) and 2,4-diaminobutyric acid (DBU, 4.5 mL, 30 mmol) in 15 mLof DCM. The mixture was heated at 40° C. for 16 hours. The mixture wasdiluted with 60 mL of DCM and 40 mL of water, then neutralized to pH 7with conc. HCl. The DCM extract was collected, washed with 0.2M HCl (60mL), then with brine (60 mL), dried over Na2SO4, and evaporated to give7.47 g of Boc protected sulfonamide. This material was suspended in 40mL of methanol, then 200 mL of 6N HCl/isopropanol was added and themixture was stirred for 2 hours. The solvent was evaporated undervacuum, 100 mL of ether was then added. The precipitate was collected byfiltration and dried to give compound 3 as HCl salt (5.93 g, 96%); MSm/z 269.1 (M+H).

Preparation of Compound 5:

To a solution of compound 4 (1.09 g, 1.6 mmol) in 10 mL ofN,N-Dimethylformamide (DMF) was added 2-(IH-7-azabenzotriazol-I-yl)-I,I,3,3-tetramethyl uranium hexafluorophosphate (HATU, 0.61 g, 1.6 mmol),diisopropylethylamine (DIEA, 0.56 mL), and compound 3 (0.49 g, 1.6 mmol)in that order. The mixture was stirred for 1 hour and diluted with 100mL of water and 4 mL of acetic acid. The precipitate was collected byfiltration, dried under vacuum and added to 10 mL of 4M HCl/dioxane.After 30 min, 200 mL of ether was added and insoluble precipitate wascollected and purified by HPLC to give compound 5 as tetrahydrofuransalt (TFA, 1.3 g, 88%); MS m/z 835.5 (M+H). Compound 5 is referred to asduostatin-3 throughout the manuscript.

Preparation of Compound 7:

To a solution of compound 5 (500 mg, 0.527 mmol) in 5 mL of DMF wasadded compound 6 (483 mg, 0.631 mmol), N-Hydroxybenzotriazole (HOBt, 40mg, 0.296 mmol), and DIEA (0.27 mL). The mixture was stirred for 16hours after which 0.4 mL of piperidine was added. After 1 hour, themixture was diluted with 100 mL of ether and the precipitate wascollected and dried to give compound 7 as HCl salt (640 mg, 95%); MS m/z1240.7 (M+H).

Preparation of Compound 9

To a solution of compound 8 (219 mg, 0.62 mmol) in 5 mL of DMF was addedHATU (236 mg, 0.62 mmol), DIEA (0.15 mL), and compound 7 (316 mg, 1.6mmol), respectively. After 1 hour, 0.2 mL of piperidine was added andthe mixture was stirred for 30 min, then purified by HPLC to givecompound 9 as TFA salt (235 mg, 64%); MS m/z 1353.8 (M+H).

Preparation of Compound 11:

To a solution of compound 9 (235 mg, 0.16 mmol) in 2 mL of methanol and1 mL of water was added a solution of dialdehyde 10 (1.6 mL of 0.3M iniPrOH) and NaCNBH3 (180 mg, 2.85 mmol). The mixture was stirred for 2hours at RT, and then purified by HPLC giving rise to compound 11 as TFAsalt (126 mg, 50%); MS m/z 1465.8 (M+H)

Generation of AXL-Specific Antibody-Drug Conjugates (ADC).

Purified AXL antibodies IgG1-AXL-148, IgG1-AXL-183 and IgG1-AXL-726 aswell as the negative control antibody IgG1-b12 were conjugated withDuostatin-3 by Concortis Biosystems, Inc. (San Diego, Calif.) throughcovalent conjugation using the K-lock AV1-valine-citruline (vc) linker[58], [148], and [149].

The anti-AXL antibody drug conjugates were subsequently analyzed forconcentration (by absorbance at 280 nm), the drug to antibody ratio (the‘DAR’) by reverse phase chromatography (RP-HPLC) and hydrophobicinteraction chromatography (HIC), the amount of unconjugated drug (byreverse phase chromatography), the percentage aggregation (bysize-exclusion chromatography, SEC-HPLC) and the endotoxin levels (byLAL). The results were as follows (Table 2):

TABLE 2 IgG1-AXL-148- IgG1-AXL-183- IgG1-AXL-726- IgG1-b12- vcDuostatin3vcDuostatin3 vcDuostatin3 vcDuostatin3 Concentration 6.57 3.40 5.93 3.36(mg/mL) DAR by HIC-HPLC 1.71 1.79 1.77 2.05 % unconjugated drug 6.674.16 5.38 4.19 % aggregate by SEC-HPLC 3.71% 3.35 3.42 1.75

Example 2—Binding Characteristics of AXL Antibodies

Binding affinity of AXL antibodies

The affinities of the panel of 9 anti-AXL antibodies as well as 3variants of these antibodies with single amino acid mutations in thevariable domains (IgG1-AXL-154-M103L, IgG1-AXL-183-N52Q,IgG1-AXL-726-M101L), were determined.

Affinities were determined using Bio-Layer Interferometry on a ForteBioOctetRED384. Anti-human Fc Capture (AHC) biosensors (ForteBio,Portsmouth, UK; cat no. 18-5064) were loaded for 150 s with hIgG (1μg/mL) aiming at a loading response of 1 nm. After a baseline (150 s)the association (1000 s) and dissociation (2000 s) of AXLECDHis (asdescribed in Example 1) was determined, using a concentration range of10 μg/mL-0.16 μg/mL (218 nM-3 nM) with 2-fold dilution steps. Forcalculations, the theoretical molecular mass of AXLECDHis based on theamino acid sequence was used, i.e. 46 kDa. Experiments were carried outon an OctetRED384, while shaking at 1000 rpm and at 30° C. Each antibodywas tested in three independent experiments.

Data was analyzed with ForteBio Data Analysis Software v7.0.3.1, usingthe 1:1 model and a global full fit with 1000 s association time and1000 s dissociation time unless stated otherwise. A dissociation time of1000 s (instead of the 2000 s dissociation time that was acquired) wasused since this resulted in better fits. For antibody IgG1-AXL-154 andIgG1-AXL-154-M103L a dissociation time of 500 s was used. ForIgG1-AXL-012 and IgG1-AXL-094 dissociation times of 200 s were used.Data traces were corrected by subtraction of a reference curve (antibodywithout AXLECDHis), the Y-axis was aligned to the last 5 s of thebaseline, and interstep correction as well as Savitzky-Golay filteringwas applied.

The affinities (K_(D)) of the anti-AXL antibodies ranged from 0.3*10⁻⁹Mto 63*10⁻⁹M (Table 3). For mutant IgG1-AXL-183-N52Q the K_(D) was lowerthan for wild-type IgG1-AXL-183, due to an approximately 2.5-fold higherdissociation rate. The observed kinetics of the other two mutants weresimilar to the kinetics of the wild-type IgGs.

TABLE 3 Binding affinity (OCTET) KD Kon Kdis Antibody (M) (1/Ms) (1/s)IgG1-AXL-107   16*10⁻⁹ 2.8*10⁵ 4.1*10⁻³ IgG1-AXL-148   20*10⁻⁹ 2.3*10⁵4.4*10⁻³ IgG1-AXL-154  7.2*10⁻⁹ 2.6*10⁵ 1.9*10⁻³ IgG1-AXL-154-M103L 7.8*10⁻⁹ 2.7*10⁵ 2.0*10⁻³ IgG1-AXL-171   17*10⁻⁹ 1.1*10⁵ 1.8*10⁻³IgG1-AXL-183 10.2*10⁻⁹ 4.1*10⁴ 4.2*10⁻⁴ IgG1-AXL-183-N52Q   24*10⁻⁹4.2*10⁴ 1.0*10⁻³ IgG1-AXL-613  1.5*10⁻⁹ 5.4*10⁵ 8.0*10⁻⁴ IgG1-AXL-726 0.6*10⁻⁹ 2.4*10⁵ 1.3*10⁻⁴ IgG1-AXL-726-M101L  0.3*10⁻⁹ 2.1*10⁵ 6.9*10⁻⁵IgG1-AXL-733   63*10⁻⁹ 1.6*10⁵ 9.7*10⁻³Binding of AXL Antibodies to Human, Mouse and Cynomolgus AXL

HEK293T cells were transiently transfected with expression constructsfor full length human AXL, human AXL with a cynomolgus monkeyextracellular domain (ECD) or human AXL with a mouse ECD (see Example1). Binding of HuMab-AXL antibodies to these cells was evaluated by flowcytometry. Transfected HEK293 cells were incubated with serial dilutionsof AXL-antibodies (final concentration range 0.0024-10 μg/mL) for 30minutes at 4° C. After washing three times in PBS/0.1% BSA/0.02% azide,cells were incubated with R-Phycoerythrin (PE)-conjugatedgoat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories, Inc.,West Grove, Pa.; cat. No. 109-116-098) diluted 1/100 in PBS/0.1%BSA/0.02% azide (final volume 100 μL). Next, cells were washed twice inPBS/0.1% BSA/0.02% azide, resuspended in 120 μL PBS/0.1% BSA/0.02% azideand analyzed on a FACS Cantoll (BD Biosciences).

Binding curves were analyzed using non-linear regression (sigmoidaldose-response with variable slope) using GraphPad Prism V5.04 software(GraphPad Software, San Diego, Calif., USA).

FIG. 1A shows that the HuMab-AXL antibodies showed dose-dependentbinding to the HEK293 cells expressing human AXL-ECD. Furthermore,HuMab-AXL antibodies recognized AXL with a cynomolgus monkey ECD, withEC₅₀ values in the same range as for fully human AXL (FIG. 1B). Incontrast, binding of HuMabs to AXL with a mouse ECD was low(IgG1-AXL-107, IgG1-AXL-154, IgG1-AXL-154-M103L, IgG1-AXL-733,IgG1-AXL-183, IgG1-AXL-183-N52Q) or not detectable (IgG1-AXL-171,IgG1-AXL-613, IgG1-AXL-726, IgG1-AXL-726-M101L, IgG1-AXL-148; FIG. 1C).As expected, the negative control antibody IgG1-b12 showed (FIG. 1) nobinding to cells expressing any of the AXL variants. Table 4 shows theEC₅₀ values and standard deviations for binding of the anti-AXLantibodies to human AXL or human AXL with a cynomolgus AXL ECD(determined in at least 3 experiments). EC₅₀ values for binding to humanAXL with a mouse AXL ECD could not be determined to very low or absentbinding.

TABLE 4 Binding EC50 (μg/mL) human AXL cynomolgus AXL Antibody Average(s.d.) Average (s.d.) IgG1-AXL-107 0.050 (0.004) 0.149 (0.021)IgG1-AXL-154 0.105 (0.003) 0.160 (0.027) IgG1-AXL-154-M103L 0.110(0.038) 0.161 (0.042) IgG1-AXL-171 0.073 (0.023) 0.157 (0.057)IgG1-AXL-613 0.040 (0.023) 0.146 (0.023) IgG1-AXL-726 0.288 (0.206)0.349 (0.160) IgG1-AXL-726-M101L 0.184 (0.117) 0.250 (0.066)IgG1-AXL-733 0.176 (0.094) 0.254 (0.114) IgG1-AXL-148 0.094 (0.059)0.152 (0.080) IgG1-AXL-183 0.526 (0.177) 0.309 (0.086) IgG1-AXL-183-N52Q0.350 (0.206) 0.324 (0.121)Competition Between AXL Antibodies and Gas6 for AXL Binding

It was tested whether the AXL ligand Gas6 interfered with binding of theAXL antibodies to AXL. Therefore, AXL-positive A431 cells were incubatedfor 15 minutes at 4° C. with 10 μg/mL recombinant human Gas6 (R&DSystems, Abingdon, UK; cat. No. 885-GS). Subsequently, serial dilutionsof AXL antibodies were prepared (final concentration range 0.014-10μg/mL), added to the cells and incubated for 30 minutes at 4° C. Afterwashing three times in PBS/0.1% BSA/0.02% azide, cells were incubated in100 μL with secondary antibody at 4° C. for 30 min in the dark. As asecondary antibody binding the Fc region, R-Phycoerythrin(PE)-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.; cat. No. 109-116-098) diluted 1/100in PBS/0.1% BSA/0.02% azide, was used. Next, cells were washed twice inPBS/0.1% BSA/0.02% azide, resuspended in 120 μL PBS/0.1% BSA/0.02% azideand analyzed on a FACS Cantoll (BD Biosciences).

Alternatively, A431 cells were pre-incubated with 10 μg/mL AXLantibodies (15 minutes, 4° C.) to assess if the AXL ligand Gas6 couldstill bind in presence of AXL antibodies. After antibody pre-incubation,serial dilutions of recombinant human Gas6 (R&D Systems, Abingdon, UK;cat. No. 885-GS) were added to the cells at final concentrations of0.001-20 μg/mL and incubated for 30 minutes at 4° C. After washing threetimes in PBS/0.1% BSA/0.02% azide, cells were incubated with mouseanti-Gas6 IgG2a (R&D Systems; cat no. MAB885) at 4° C. for 30 min. Afterwashing three times in PBS/0.1% BSA/0.02% azide, cells were incubatedwith FITC-labelled goat anti-mouse IgG (Dako, Heverlee, Belgium; cat no.F049702) at 4° C. for 30 min in the dark. Next, cells were washed twicein PBS/0.1% BSA/0.02% azide, resuspended in 120 μL PBS/0.1% BSA/0.02%azide and analyzed on a FACS Cantoll (BD Biosciences).

Binding curves were analyzed using non-linear regression (sigmoidaldose-response with variable slope) using GraphPad Prism V5.04 software(GraphPad Software, San Diego, Calif., USA).

In experiments (n=3) in which A431 cells were pre-incubated with Gas6,the maximal binding values of anti-AXL antibodies was comparable toantibody binding in absence of Gas6 (maximal binding after Gas6pre-incubation was 90-108% of binding without Gas6 pre-incubation)(Table 4). The EC₅₀ values for AXL antibody binding with or without Gas6pre-incubation were in the same range, or somewhat enhanced after Gas6pre-incubation (Table 5).

The binding of control AXL antibody YW327.652 to A431 cells was greatlyreduced in the presence of Gas6 compared to binding without Gas. Maximalbinding of YW327.652 in the presence of Gas6 was 19% of binding withoutGas6, and the EC50 value for binding to A431 cells was 21-fold higherwhen cells had been pre-incubated with Gas6.

In experiments in which A431 cells were pre-incubated with anti-AXLantibodies, Gas6 binding was evaluated (n=3). Binding of Gas6 to A431cells was similar with or without pre-incubation with HuMab-AXLantibodies. Average EC50 concentrations of Gas6 binding when cells werepre-incubated with HuMabs (0.34-0.83 μg/mL) and maximal Gas6 bindingwere similar to Gas6 binding in the presence of negative controlantibody b12 (EC50 concentration: 0.40 μg/mL; 95-115% of Gas6 binding inthe presence of the b12 control antibody). The binding of Gas6 to A431cells was greatly reduced in the presence of control AXL antibodyYW327.652 compared to pre-incubation with b12 (the EC50 concentrationwas 14-fold higher). Maximal binding of Gas6 in the presence of controlantibody YW327.652 was 17% of binding in the presence of negativecontrol antibody b12.

TABLE 5 Antibody binding to A431 cells Maximal Gas6 binding to A431cells binding in Maximal binding presence of EC50 in in presence of EC50w/o EC50 in Gas6 presence AXL antibodies Gas6 presence of binding in ofAXL (% of binding in EC50 Gas6 absence of antibodies prescence of(μg/mL) (μg/mL) Gas6) (μg/mL) control antibody) Antibody mean (s.d.)mean (s.d.) mean (s.d.) mean (s.d.) mean (s.d.) IgG1-AXL-107 0.16 (0.17)0.94 (1.18)  91 (5) 0.78 (0.54)  96 (8) IgG1-AXL-148 0.11 (0.13) 0.20(0.30)  93 (5) 0.73 (0.52) 106 (7) IgG1-AXL-154 0.42 (0.55) 0.76 (0.78) 99 (13) 0.44 (0.28)  95 (10) IgG1-AXL-171 0.18 (0.21) 0.32 (0.40)  95(5) 0.69 (0.42) 108 (5) IgG1-AXL-183 0.69 (0.72) 1.19 (1.11)  90 (19)0.34 (0.13) 115 (8) IgG1-AXL-511 0.12 (0.11) 0.30 (0.31)  93 (15) 0.74(0.44) 113 (6) IgG1-AXL-613 0.09 (0.09) 0.10 (0.10) 108 (22) 0.57 (0.36)100 (11) IgG1-AXL-726 0.32 (0.35) 0.55 (0.69)  97 (10) 0.77 (0.58)  98(10) IgG1-AXL-733 0.49 (0.51) 0.62 (0.23)  93 (5) 0.83 (0.54)  96 (5)YW327.6S2 0.09 (0.09) 1.90 (1.04)*  41 (24) 5.53 (7.09)*  17 (10) b12n.a.^(a) n.a. n.a. 0.40 (0.11) 100 ^(a)n.a., not applicable *EC50 valuesless accurate due to low binding.

Example 3—Epitope Mapping Studies Anti-AXL Antibody Panel

Determining the AXL Domain Specificity Using Human-Mouse AXL ChimericMolecules

The AXL domain specificity of the AXL antibodies was determined using apanel of human-mouse chimeric AXL mutants. Five different chimeric AXLmolecules were generated, in which either the human Ig-like domain I(Ig1), the Ig-like domain II (Ig2), the human FNIII-like domain I (FN1)or the human FNIII-like domain II domain (FN2) were replaced with theirmurine homologs.

The following codon-optimized constructs for expression of the AXLhuman-mouse chimeras were generated and expressed in HEK293F cells asdescribed in Example 1:

Homo sapiens AXL (p33-HAHs-AXL): (SEQ ID NO: 130)MAWRCPRMGRVPLAWCLALCGWACMYPYDVPDYAAPRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSNDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQE DGA Mus musculus AXL(p33-HAMm-AXL): (SEQ ID NO: 131)MAWRCPRMGRVPLAWCLALCGWACMYPYDVPDYAAHKDTQTEAGSPFVGNPGNITGARGLTGTLRCELQVQGEPPEVVWLRDGQILELADNTQTQVPLGEDWQDEWKVVSQLRISALQLSDAGEYQCMVHLEGRTFVSQPGFVGLEGLPYFLEEPEDKAVPANTPFNLSCQAQGPPEPVTLLWLQDAVPLAPVTGHSSQHSLQTPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQRPHHLHVVSRQPTELEVAWTPGLSGIYPLTHCNLQAVLSDDGVGIWLGKSDPPEDPLTLQVSVPPHQLRLEKLLPHTPYHIRISCSSSQGPSPWTHWLPVETTEGVPLGPPENVSAMRNGSQVLVRWQEPRVPLQGTLLGYRLAYRGQDTPEVLMDIGLTREVTLELRGDRPVANLTVSVTAYTSAGDGPWSLPVPLEPWRPGQGQPLHHLVSEPPPRAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQ EDGA Homo sapiensAXL - Mus musculus Ig1 domain (p33-AXL-mIg1): (SEQ ID NO: 132)MGRVPLAWWLALCCWGCAAHKDTQTEAGSPFVGNPGNITGARGLTGTLRCELQVQGEPPEVVWLRDGQILELADNTQTQVPLGEDWQDEWKVVSQLRISALQLSDAGEYQCMVHLEGRTFVSQPGFVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQEDGA Homo sapiens AXL - Mus musculusIg2 domain (p33-AXL-mIg2): (SEQ ID NO: 133)MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDKAVPANTPFNLSCQAQGPPEPVTLLWLQDAVPLAPVTGHSSQHSLQTPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQEDGA Homo sapiens AXL - Musmusculus FN1 domain (p33-AXL-mFN1): (SEQ ID NO: 134)MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQRPHHLHVVSRQPTELEVAWTPGLSGIYPLTHCNLQAVLSDDGVGIWLGKSDPPEDPLTLQVSVPPHQLRLEKLLPHTPYHIRISCSSSQGPSPWTHWLPVETTEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQEDGA Homo sapiens AXL - Musmusculus FN2 domain (p33-AXL-mFN2): (SEQ ID NO: 135)MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENVSAMRNGSQVLVRWQEPRVPLQGTLLGYRLAYRGQDTPEVLMDIGLTREVTLELRGDRPVANLTVSVTAYTSAGDGPWSLPVPLEPWRPGQGQPLHHLVSEPPPRAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQEDGA

Binding of 1 μg/mL anti-AXL antibody to the human-mouse AXL chimeras wasdetermined by flow cytometry, as described in Example 2. IgG1-b12 wasincluded as an isotype control IgG1.

All anti-AXL antibodies showed binding to human AXL (FIG. 2A), whereasbinding was abrogated or strongly reduced when the human AXL ECD wasreplaced with its murine homolog (FIG. 2B). The human-mousecross-reactive monoclonal AXL antibody YW327.6S2 was included to confirmexpression of hsAXL-mmECD.

Anti-AXL antibody 107 and 613 showed strongly reduced binding tohsAXL-mmIg1 (FIG. 2C), indicating recognition of an epitope in the AXLIg1 domain. IgG1-AXL-148 and IgG1-AXL-171 showed strongly reducedbinding to hsAXL-mmIg2 (FIG. 2D), indicating recognition of an epitopein the AXL Ig2 domain. IgG1-AXL-154, IgG1-AXL-183 and IgG1-AXL-733showed reduced binding to hsAXL-mmFN1 (FIG. 2E), indicative of a bindingepitope in the AXL FN1 domain. Finally, binding of IgG1-AXL-726 was lostin hsAXL-mmFN2 (FIG. 2F), indicating recognition of an epitope withinthe FN2 domain.

AXL domain specificity for all anti-AXL antibodies is summarized inTable 6.

TABLE 6 AXL domain AXL aa's involved in Antibody specificity bindingIgG1-AXL-107 Ig1 L121-Q129 IgG1-AXL-148 Ig2 D170-R190 IgG1-AXL-154 Fn1Q272-A287, G297-P301 IgG1-AXL-154- n.d.^(a) n.d. M103L IgG1-AXL-171 Ig2P170, T182-R190 IgG1-AXL-183 Fn1 Not resolved IgG1-AXL-183- n.d. n.d.N52Q IgG1-AXL-613 Ig1 T112-Q124 IgG1-AXL-726 Fn2 A359, R386, Q436-K439IgG1-AXL-726- n.d. n.d. M101L IgG1-AXL-733 Fn1 Not resolved 1gG1-AXL-061Ig1 197-Q124 1gG1-AXL-137 Ig1 Q57, E92-T105 YW327.6S2 Ig1 G39-D59^(a)n.d., not determinedHigh Resolution Epitope Mapping to Identify Amino Acids in the AXLExtracellular Domain Involved in Binding of AXL Antibodies

To identify amino acids in the AXL extracellular domain involved inbinding of anti-AXL antibodies, a library of AXL sequence variants wasgenerated by recombination of AXL sequences derived from species withvariable levels of homology with the human AXL sequence in theextracellular domain. Briefly, an expression plasmid encoding human AXL(Hs) was mixed with cloning plasmids encoding Mus musculus (Mm),Monodelphis domestica (Md; opossum) Anolis carolinensis (Ac; lizard) andTetraodon nigroviridis (Tn; pufferfish) AXL homologs or vice versa. Acombination of two primers specific to either the cloning or theexpression vector was used to perform a PCR amplifying the AXLextracellular domain (ECD) with abbreviated elongation time, forcingmelting and reannealing of nascent DNA replication strands during PCRcycling. Full length ECD was amplified using a nested PCR, againspecific to recombination products containing termini originating fromboth vectors.

Resulting AXL ECD PCR products were cloned into an expression vectorcreating full length AXL, and resulting plasmids were sequenced, rankedby maximal difference to the template vectors and selected to create aminimal ensemble with maximal differentiation power. Plasmids encodingAXL homologs from Hs, Mm, Md, Ac and Tn, four human/mouse chimericplasmids encoding Hs AXL with murine Ig1, Ig2, Fn1 or Fn2 domains, andthe sixteen most differentiating plasmids from the recombination librarywere transfected into HEK293-F cells according to the specificationssupplied by the manufacturer (Life technologies). FACS binding datausing 1 μg/mL anti-AXL antibodies were deconvoluted by scoring per aminoacid if mutation did (+1) or did not (−1) correlate with loss ofbinding, after which a baseline correction and normalization to a scaleof −5 to +5 was applied, resulting in an impact score per amino acidover the full ECD.

The deconvoluted binding data is summarized in Table 6 as the aminoacids involved in binding. Antibodies of which the binding site couldnot be mapped to high resolution due to a lack of recombination eventsin the proximity of the binding site, are indicated as not resolved.

Example 4—Fc-Mediated Effector Functions

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

The ability of anti-AXL antibodies to induce ADCC of A431 epidermoidcarcinoma cells was determined as explained below. As effector cells,peripheral blood mononuclear cells from healthy volunteers (UMC Utrecht,The Netherlands) were used.

Labeling of Target Cells

A431 cells were collected (5×10⁶ cells) in culture medium (RPMI 1640culture medium supplemented with 10% fetal calf serum (FSC)), to which100 μCi ⁵¹Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal,The Netherlands) had been added, and the mixture was incubated in a 37°C. water bath for 1 hour (hr) while shaking. After washing of the cells(twice in PBS, 1200 rpm, 5 min), the cells were resuspended inRPM11640/10% FSC and counted by trypan blue exclusion. Cells werediluted to a density of 1×10⁵ cells/m L.

Preparation of Effector Cells

Peripheral blood mononuclear cells (healthy volunteers, UMC Utrecht,Utrecht, The Netherlands) were isolated from 45 mL of freshly drawnheparin blood by Ficoll (Bio Whittaker; lymphocyte separation medium,cat 17-829E) according to the manufacturer's instructions. Afterresuspension of cells in RPM11640/10% FSC, cells were counted by trypanblue exclusion and diluted to a density of 1×10⁷ cells/mL.

ADCC Set Up 50 μl of ⁵¹Cr-labeled targets cells were pipetted into96-well plates, and 50 μl of antibody were added, diluted inRPM11640/10% FSC (3-fold dilutions at final concentrations range 0.01-10μg/mL). Cells were incubated (room temperature (RT), 15 min), and 50 μleffector cells were added, resulting in an effector to target ratio of100:1 (for determination of maximal lysis, 100 μl 5% Triton-X100 wasadded instead of effector cells; for determination of spontaneous lysis,50 μL target cells and 100 μL RPM11640/10% FSC were used). Cells wereincubated overnight at 37° C. and 5% CO₂. After spinning down cells(1200 rpm, 10 min), 70 μL of supernatant was harvested into micronictubes, and counted in a gamma counter. The percentage specific lysis wascalculated as follows:% specific lysis=(cpm sample−cpm target cells only)/(cpm maximallysis−cpm target cells only)wherein cpm is counts per minute.

IgG1-AXL-183-N52Q, and IgG1-AXL-733 induced 15 to 21% ADCC in A431 cellsat a concentration of 10 μg/mL (FIG. 3). IgG1-AXL-148,IgG1-AXL-726-M101L, IgG1-AXL-171, IgG1-AXL-613, IgG1-AXL-107, andIgG1-AXL-154-M103L did not induce significant ADCC in A431 cell atconcentrations up to 10 μg/mL (FIG. 3).

Example 5—Binding Characteristics of AXL Antibody-Drug Conjugates(AXL-ADCs)

HEK293T cells were transiently transfected with expression constructsfor full-length human AXL (see Example 1). Binding of anti-AXLantibodies and AXL-ADCs to these cells was evaluated by flow cytometry.Transiently transfected HEK293 cells were incubated with serialdilutions of anti-AXL antibodies or AXL-ADCs (4-fold dilutions; finalconcentration range 0.003-10 μg/mL) for 30 minutes at 4° C. Afterwashing three times in PBS/0.1% BSA/0.02% azide, cells were incubated in100 μL with secondary antibody at 4° C. for 30 min in the dark. As asecondary antibody, R-Phycoerythrin (PE)-conjugated goat-anti-human IgGF(ab′)2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.;cat. No. 109-116-098) diluted 1/100 in PBS/0.1% BSA/0.02% azide, wasused. Next, cells were washed twice in PBS/0.1% BSA/0.02% azide,resuspended in 120 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACSCantoll (BD Biosciences).

Binding curves were analyzed using non-linear regression (sigmoidaldose-response with variable slope) using GraphPad Prism V5.04 software(GraphPad Software, San Diego, Calif., USA).

FIG. 4 shows that binding of the anti-AXL antibodies to the HEK293 cellsexpressing human AXL-ECD was similar to the binding of the AXL-ADCs.

Example 6—In Vitro Cytotoxicity Induced by AXL-Specific Antibody DrugConjugates

LCLC-103H cells (human large cell lung cancer) cells were cultured inRPMI 1640 with L-Glutamine (Cambrex; cat.no. BE12-115F) supplementedwith 10% (vol/vol) heat inactivated Cosmic Calf Serum (Perbio; cat.no.SH30087.03), 2 mM L-glutamine (Cambrex; cat.no. US17-905C), 50 IU/mLpenicillin, and 50 μg/mL streptomycin (Cambrex; cat.no. DE17-603E).MDA-MB-231 cells (human breast cancer) were cultured in DMEM (Cambrex;cat.no. BE12-709F) supplemented with 10% (vol/vol) heat inactivatedCosmic Calf Serum (Perbio; cat.no. SH30087.03), 1 mM Sodium Pyruvate(Cambrex; cat.no. 13E13-115E), 2 mM L-glutamine (Cambrex; cat.no.US17-905C), 100 μM MEM NEAA (Invitrogen; cat.no. 11140), 50 IU/mLpenicillin, and 50 μg/mL streptomycin (Cambrex; cat.no. DE17-603E). Thecell lines were maintained at 37° C. in a 5% (vol/vol) CO2 humidifiedincubator. LCLC-103H and MDA-MB-231 cells were cultured to nearconfluency, after which cells were trypsinized, resuspended in culturemedium and passed through a cell strainer (BD Falcon, cat.no. 352340) toobtain a single cell suspension. 1×10³ cells were seeded in each well ofa 96-well culture plate, and cells were incubated for 30 min at roomtemperature and subsequently for 5 hrs at 37° C., 5% CO2 to allowadherence to the plate.

Serial dilutions (4-fold; final concentrations ranging from 0.00015 to10 μg/mL) of AXL antibody drug conjugates (AXL-ADCs; see Example 1) wereprepared in culture medium and added to the plates. Incubation of cellswith 1 μM staurosporin (#56942-200, Sigma) was used as reference for100% tumor cell kill. Untreated cells were used as reference for 0%tumor cell kill. Plates were incubated for 5 days at 37° C., 5% CO2.Next, CellTiter-Glo Reagent (Promega; cat.no. G7571) was added to thewells (20 μL per well) and plates were incubated for 1.5 hours at 37°C., 5% CO2. Subsequently, 180 μL per well was transferred to white96-well Optiplate™ plates (PerkinElmer, Waltham, Mass.; cat.no.6005299), which were incubated for 30 min at room temperature. Finally,luminescence was measured on an EnVision multiplate reader (Envision,Perkin Elmer).

AXL-ADCs IgG1-AXL-148-vcDuo3, IgG1-AXL-183-vcDuo3, andIgG1-AXL-726-vcDuo3 induced cytotoxicity in LCLC-103H cells, with 1050values between 0.01 and 0.06 μg/mL, as shown in FIG. 5A. Similarly, FIG.5B shows that these AXL-ADCs induced cytoxicity of MDA-MB-231 cells withIC50 values between 0.005 and 0.015 μg/mL.

Example 7—Antibody VH and VL Variants that Allow Binding to AXL

Protein sequences of the VH and VL regions of the anti-AXL antibodypanel (described in Example 1) were aligned and compared for AXL bindingto identify critical or permissive changes of amino acid residues in theVH or VL regions. Therefore, antibodies with identical VH or VL regionswere grouped and compared for binding to human AXL and differences in VLor VH sequences, respectively. Binding to human AXL transientlyexpressed by HEK-293F cells was assessed in the homogeneous antigenspecific screening assay as described in Example 1. Numbering of aminoacid positions for the alignments done in the present example was donebased on the sequences put forth in FIG. 6, i.e. the first amino acid inthe shown sequence was numbered as position ‘1’, the second as position‘2’, etc.

First, antibodies with identical VL sequences were grouped.

IgG1-AXL-148 and IgG1-AXL-140 were found to have an identical VLsequence, and showed 1 amino acid difference in the HC CDR3 region (Ffor I at amino acid position 109; FIG. 6A). Both antibodies bound tohuman AXL (Table 7), indicating that the amino acid at position 109 isnot essential for antibody binding, assuming that a mutation identifiedin the CDR2 region (G for A at the amino acid position 56) does notcompensate for loss of binding (FIG. 6A).

IgG1-AXL-726 and IgG1-AXL-187 were found to have an identical VLsequence and both antibodies bound to human AXL (Table 7). Two aminoacid residue changes in the HC CDR3 region (R for S at position 97 and Afor T at position 105; FIG. 6B) were allowed without losing binding,assuming that mutations identified in the CDR1 (Y for H at position 32)and/or in the framework regions (P3Q, V241, Y25D, T86A and T117A) do notcompensate for loss of binding (FIG. 6B).

IgG1-AXL-171, IgG1-AXL-172 and IgG1-AXL-181 were found to have anidentical VL sequence and all antibodies bound to human AXL (Table 7).The CDR3 regions of these three antibodies were identical, but an aminoacid residue change in the HC CDR1 (S for N at position 31) or theframework region (H for Q at position 82) was allowed without losingbinding (FIG. 6C).

IgG1-AXL-613, IgG1-AXL-608-01, IgG1-AXL-610-01 and IgG1-AXL-620-06 werefound to have an identical VL sequence, and showed one amino aciddifference in the HC CDR3 region (N for D at amino acid position 101;FIG. 6D). All antibodies bound to human AXL (Table 7), indicating thatthe amino acid at position 101 is not essential, assuming that mutationsidentified in the HC CDR2 (V for A at position 58) and/or in theframework regions (N35S, M37V, A61V, L70I, S88A) do not compensate forloss of binding (FIG. 6D).

Next, antibodies with identical VH sequences were grouped.

IgG1-AXL-613 and IgG1-AXL-613-08 were found to have an identical VHsequence, and showed five amino acid differences in the CDR3 region ofthe LC (RSNWL for YGSSY at positions 92 to 96; FIG. 6E). Both antibodiesbound to human AXL (Table 7), indicating that the variation of aminoacid at positions 92 to 96 are allowed and do not affect antibodybinding, assuming that mutations identified in the CDR1 (deletion of theS at position 30), CDR2 (G51D), and/or in the framework regions (G9A,S54N, R78S, Q100G, L104V) do not compensate for loss of binding (FIG.6E).

TABLE 7 EC50 Maximal binding Antibody (μg/mL) (Arbitrary units)IgG1-AXL-140 0.0026 2889 IgG1-AXL-148 0.0036 3499 IgG1-AXL-171 0.0032575 IgG1-AXL-172 0.0055 5378 IgG1-AXL-181 0.008 3598 IgG1-AXL-1870.0065 2563 IgG1-AXL-608-01 0.0035 3318 IgG1-AXL-610-01 0.0023 2947IgG1-AXL-613 0.0072 5211 IgG1-AXL-613-08 0.0242 2209 IgG1-AXL-620-060.0034 4352 IgG1-AXL-726 0.0471 3154

Example 8—In Vitro Cytotoxicity Induced by MMAE-Conjugated AXLAntibodies

Conjugation of MMAE to Anti-AXL Antibodies

Anti-AXL antibodies were purified by Protein A chromatography accordingto standard procedures and conjugated to vcMMAE. The drug-linker vcMMAEwas alkylated to the cysteines of the reduced antibodies according toprocedures described in the literature (see [150], [151], and [152]).The reaction was quenched by the addition of an excess ofN-acetylcysteine. Any residual unconjugated drug was removed bypurification and the final anti-AXL antibody drug conjugates wereformulated in PBS. The anti-AXL antibody drug conjugates weresubsequently analyzed for concentration (by absorbance at 280 nm), thedrug to antibody ratio (DAR) by reverse phase chromatography (RP-HPLC)and hydrophobic interaction chromatography (HIC), the amount ofunconjugated drug (by reverse phase chromatography), the percentageaggregation (by size-exclusion chromatography, SEC-HPLC) and theendotoxin levels (by LAL). The results are shown below in Table 8.

TABLE 8 Overview of different characteristics of the antibody-drugconjugates. ADC IgG1- IgG1- IgG1- IgG1- IgG1- AXL- IgG1- AXL- IgG1-IgG1- AXL- IgG1- AXL- AXL- 154- AXL- 183- AXL- AXL- 726- AXL- IgG1-Assay 107 148 M103L 171 N52Q 511 613 M101L 733 b12 Concentration 7.189.63 6.57 3.69 6.71 5.77 6.17 7.37 7.71 1.58 (mg/mL) DAR by HIC 3.973.96 3.71 3.65 3.92 3.87 4.23 4.12 4.08 4.00 % unconjugated 4.68 5.586.13 7.11 8.68 8.35 5.13 4.99 3.74 1.89 antibody % aggregate by 6.3 2.282.9 3.3 5.2 5.1 6.4 4.0 3.5 2.5 SEC-HPLC Endotoxin 2.3 1.2 2.6 3.1 5.94.5 2.0 3.6 7.6 11.5 (EU/mg)Cell Culture

LCLC

103H cells (human large cell lung cancer) and A431 cells (DMSZ,Braunschweig, Germany) were cultured in RPMI 1640 with L-Glutamine(Cambrex; cat.no. BE12-115F) supplemented with 10% (vol/vol) heatinactivated Cosmic Calf Serum (Perbio; cat.no. SH30087.03), 2 mML-glutamine (Cambrex; cat.no. US17-905C), 50 IU/mL penicillin, and 50μg/mL streptomycin (Cambrex; cat.no. DE17-603E). MDA-MB231 cells werecultured in DMEM with high glucose and HEPES (Lonza #BE12-709F), DonorBovine Serum with Iron (Life Technologies #10371-029), 2 mM L-glutamine(Lonza #BE17-605E), 1 mM Sodium Pyruvate (Lonza #BE13-115E), and MEMNon-Essential Amino Acids Solution (Life Technologies #11140). The celllines were maintained at 37° C. in a 5% (vol/vol) CO₂ humidifiedincubator. LCLC-103H, A431 and MDA-MB231 cells were cultured to nearconfluency, after which cells were trypsinized, resuspended in culturemedium and passed through a cell strainer (BD Falcon, cat.no. 352340) toobtain a single cell suspension. 1×10³ cells were seeded in each well ofa 96-well culture plate, and cells were incubated for 30 min at roomtemperature and subsequently for 5 hrs at 37° C., 5% CO₂ to allowadherence to the plate.

Cytotoxicity Assay

Serial dilutions (final concentrations ranging from 0.00015 to 10 μg/mL)of MMAE-conjugated AXL-antibodies were prepared in culture medium andadded to the plates. Incubation of cells with 1 μM staurosporin(#56942-200, Sigma) was used as reference for 100% tumor cell kill.Untreated cells were used as reference for 100% cell growth. Plates wereincubated for 5 days at 37° C., 5% CO₂. Next, CellTiter-Glo Reagent(Promega; cat.no. G7571) was added to the wells (20 μL per well) andplates were incubated for 1.5 hours at 37° C., 5% CO₂. Subsequently, 180μL per well was transferred to white 96-well Optiplate™ plates(PerkinElmer, Waltham, Mass.; cat.no. 6005299), which were incubated for30 min at room temperature. Finally, luminescence was measured on anEnVision multiplate reader (Envision, Perkin Elmer).

MMAE-conjugated AXL-antibodies induced 50% cell kill in LCLC-103H cellsat concentrations between 0.004 and 0.219 μg/mL as shown in Table 9a andFIG. 7.

Similarly, AXL-ADCs efficiently induced cytotoxicity in A431 cells(Table 9b) and FIG. 15A) and MDA-MB231 cells (Table 9b and FIG. 15B).

TABLE 9a Cytotoxicity of MMAE-conjugated-AXL-antibodies in LCLC-103Hcells (EC50 values) EC50 ADC (μg/mL) IgG1-AXL-613-vcMMAE 0.004IgG1-AXL-148-vcMMAE 0.012 IgG1-AXL-171-vcMMAE 0.018IgG1-AXL-726-M101L-vcMMAE 0.018 IgG1-AXL-107-vcMMAE 0.022IgG1-AXL-511-vcMMAE 0.032 IgG1-AXL-154-M103L-vcMMAE 0.044IgG1-AXL-183-N52Q-vcMMAE 0.113 IgG1-AXL-733-vcMMAE 0.219

TABLE 9b Cytotoxicity of MMAE-conjugated AXL antibodies in A431 andMDA-MB-231 cells (EC50 values). EC50 (μg/mL) A431 MDA-MB231 (n = 3) (n =2) ADC Mean s.d. Mean s.d. IgG1-AXL-107-vcMMAE 0.154 0.066 0.037 0.005IgG1-AXL-148-vcMMAE 0.070 0.013 0.012 0.004 IgG1-AXL-154-M103L-vcMMAE0.719 0.091 0.396 0.195 IgG1-AXL-171-vcMMAE 0.206 0.074 0.035 0.006IgG1-AXL-183-N52Q-vcMMAE 1.157 0.160 0.139 0.028 IgG1-AXL-511-vcMMAE0.093 0.020 0.052 0.003 IgG1-AXL-613-vcMMAE 0.109 0.078 0.005 0.001IgG1-AXL-726-M101L-vcMMAE 0.270 0.157 0.022 0.002 IgG1-AXL-733-vcMMAE1.253 0.228 0.881 0.182

Example 9—Therapeutic Treatment of LCLC-103H Tumor Xenografts in SCIDMice with MMAE-Conjugated Anti-AXL Antibodies

The in vivo efficacy of MMAE-conjugated anti-AXL antibodies wasdetermined in established subcutaneous (SC) LCLC-103H xenograft tumorsin SCID mice. 5×10⁶ LCLC-103H (large cell lung carcinoma) tumor cells(obtained from Leibniz-Institut DSMZ-Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSMZ)) in 200 μL PBS wereinjected subcutaneously in the right flank of female SCID mice. Starting14-21 days after tumor cell inoculation, when the average tumor sizewas >100-200 mm³ and distinct tumor growth was observed, a singleinjection with 1 mg/kg (20 μg/mouse) IgG1-AXL-vcMMAE antibodies (asdescribed in Supplementary Example 1) or control (unconjugated IgG1-b12)was given intraperitoneally (100 μL/mouse). Tumor volume was determinedat least two times per week. Tumor volumes (mm³) were calculated fromcaliper (PLEXX) measurements as: 0.52×(length)×(width)².

The panel of anti-AXL-vcMMAE antibodies showed a broad range ofanti-tumor activity in established SC LCLC-103H tumors (FIG. 8). ClonesIgG1-AXL-733-vcMMAE, IgG1-AXL-107-vcMMAE and IgG1-AXL-148-vcMMAE inducedtumor regression, clones AXL-171-vcMMAE, IgG1-AXL-511-vcMMAE andIgG1-AXL-613-vcMMAE induced tumor growth inhibition, and clonesIgG1-AXL-154-M103L-vcMMAE, IgG1-AXL-183-N52Q-vcMMAE, andIgG1-AXL-726-M101L-vcMMAE showed no or only minor tumor growthinhibition.

Statistical analysis on the last day that all groups were intact (day30) using One Way ANOVA (Dunnett's multiple comparisons test versuscontrol IgG1-b12) indicated a highly significant difference (p<0.0001)in tumor volume between IgG1-b12 versus IgG1-AXL-733-vcMMAE,IgG1-AXL-107-vcMMAE and IgG1-AXL-148-vcMMAE. Treatment with these clonesled in some mice within these groups to complete tumor reduction.Treatment with clones IgG1-AXL-171-vcMMAE, IgG1-AXL-511-vcMMAE andIgG1-AXL-613-vcMMAE also showed significant tumor growth inhibitioncompared to IgG1-b12, but the differences were less pronounced (p<0.05to p<0.001). The tumor growth of mice treated with clonesIgG1-AXL-154-M103L-vcMMAE, IgG1-AXL-183-N52Q-vcMMAE, andIgG1-AXL-726-M101L-vcMMAE was not significant affected compared to theIgG1-b12 control.

Anti-tumor activity of anti-AXL-vcMMAE antibodies was observed invarious other in vivo tumor models. In two cell line-derived xenograftmodels (A431; epidermoid adenocarcinoma, and MDA-MB-231; breast cancer)anti-AXL-vcMMAE antibodies induced tumor growth inhibition, and tumorregression was induced by anti-AXL-vcMMAE antibodies in twopatient-derived xenograft models from patients with pancreas cancer andcervical cancer.

Example 10—Anti-Tumor Efficacy of AXL-ADCs in a Pancreas CancerPatient-Derived Xenograft (PDX) Model with Heterogeneous TargetExpression

The anti-tumor activity of IgG1-AXL-107-vcMMAE, IgG1-AXL-148-vcMMAE, andIgG1-AXL-733-vcMMAE was determined in the PAXF1657 pancreas cancer PDXmodel (experiments performed by Oncotest, Freiburg, Germany). Humanpancreas tumor tissue was subcutaneously implanted in the left flank of5-7 weeks old female NMRI nu/nu mice. Randomization of animals wasperformed as follows: animals bearing a tumor with a volume between50-250 mm³, preferably 80-200 mm³, were distributed in 7 experimentalgroups (8 animals per group), considering a comparable median and meanof group tumor volume. At day of randomization (day 0), the 3 ADCs weredosed intravenously (i.v.) at either 4 mg/kg or 2 mg/kg, and the controlgroup received a single dose of IgG1-b12 (4 mg/kg). Tumor volumes (mm³)were monitored twice weekly and were calculated from caliper (PLEXX)measurements as: 0.52×(length)×(width)².

Staining of PAXF1657 tumors was performed with standardimmunohistochemistry techniques. Briefly, frozen tissues were fixatedwith acetone for 10 minutes and endogenous peroxidase was exhaustedusing hydrogen peroxidase. Subsequently, tissue sections were blockedwith normal mouse serum and staining was performed by incubation with 5μg/mL of a pool of 5 IgG1-AXL antibodies (IgG1-AXL-061, IgG1-AXL-137,IgG1-AXL-148, IgG1-AXL-183, IgG1-AXL-726). After incubation with thesecondary, horseradish peroxidase (HRP) conjugated antibody, HRP wasvisualized with amino-ethyl carbazole (AEC; resulting in a red color).Each slide was counterstained with hematoxylin (blue) to identify nucleiand coverslipped in glycergel. Immunostained tissue slices weredigitized on manual Zeiss microscope (AxioSkop) at 10× and 40×magnifications.

FIG. 9 shows heterogeneous AXL expression in PAXF1657 tumors. Whereasstrong AXL staining is observed in some tumor cells, other cells do notshow AXL staining. In black and white photo the AXL staining appears asdark grey. Hematoxylin staining (nuclei) appears as light grey.

FIG. 10A shows that treatment of mice with 2 mg/kg IgG1-AXL-107-vcMMAE,IgG1-AXL-148-vcMMAE and IgG1-AXL-733-vcMMAE significantly reduced thegrowth of PAXF1657 tumors compared to the control group. At a dose of 4mg/kg IgG1-AXL-107-vcMMAE, IgG1-AXL-148-vcMMAE and IgG1-AXL-733-vcMMAEinduced tumor regression of PAXF1657 tumors. On day 14 after treatment,the average tumor size in mice that had been treated with 2 mg/kg or 4mg/kg IgG1-AXL-107-MMAE, IgG1-AXL-148-MMAE or IgG1-AXL-733-MMAE wassignificantly smaller than in mice that had been treated with an isotypecontrol IgG (IgG1-b12) (p<0.001; Tukey's multiple comparison test).

Treatment of mice with unconjugated IgG1-AXL-148 did not result inanti-tumor activity in the PAXF1657 model (FIG. 10B). ConjugatedIgG1-AXL-148-vcMMAE, however, induced dose-dependent antitumor activityin this model (FIG. 10B), illustrating that the therapeutic capacity ofAXL-ADCs is dependent on the cytotoxic activity of MMAE.

Moreover, treatment of mice with the untargeted ADC IgG1-b12-vcMMAE didnot show anti-tumor activity in the PAXF1657 model (FIG. 10C),illustrating that the therapeutic capacity of AXL-ADCs also depends onspecific target binding.

Example 11—AXL Antibodies Binding to the Ig1 Domain

The AXL domain specificity of AXL antibodies IgG1-AXL-061, IgG1-AXL-107,IgG1-AXL-137, and IgG1-AXL-613 was determined using a panel ofhuman-mouse chimeric AXL mutants. The human-mouse cross-reactivemonoclonal AXL antibody YW327.6S2 was included to confirm expression ofhsAXL-mmECD. IgG1-b12 was included as isotype control antibody. Fivedifferent chimeric AXL molecules were generated and expressed in HEK293Fas described in Example 3. In brief, the human Ig-like domain I (Ig1),the Ig-like domain II (Ig2), the human FNIII-like domain I (FN1) or thehuman FNIII-like domain II domain (FN2) were replaced with their murinehomologs. Binding of 1 μg/mL anti-AXL antibody to the human-mouse AXLchimeras was determined by flow cytometry, as described in Example 2.

All anti-AXL antibodies showed binding to human AXL (FIG. 11A), whereasbinding was abrogated when the human AXL ECD was replaced with itsmurine homolog (FIG. 11B). As expected, the human-mouse cross-reactivemonoclonal AXL antibody YW327.6S2 showed binding to hsAXL-mmECD,confirming proper expression of hsAXL-mmECD.

AXL antibodies IgG1-AXL-061, IgG1-AXL-107, IgG1-AXL-137, andIgG1-AXL-613 showed strongly reduced binding to hsAXL-mmIg1 (FIG. 11C),illustrating recognition of an epitope in the AXL Ig1 domain. In linewith this, binding of IgG1-AXL-061, IgG1-AXL-107, IgG1-AXL-137, andIgG1-AXL-613 to hsAXL-mmIg2 (FIG. 11D), hsAXL-mmFN1 (FIG. 11E) orhsAXL-mmFN2 (FIG. 11F) was not affected. The human-mouse cross-reactivemonoclonal AXL antibody YW327.6S2 showed binding to all chimeric AXLvariants, confirming proper expression of these proteins.

Example 12—AXL Antibodies IgG1-AXL-107 and IgG1-AXL-613 Bind to the Ig1Domain but do not Compete with Gas6 Binding

It was tested whether the binding of the AXL antibodies IgG1-AXL-061,IgG1-AXL-107, IgG1-AXL-137, or IgG1-AXL-613 interfered with binding ofAXL ligand Gas6 to AXL. Therefore, binding of Gas6 to A431 cells thathad been pre-incubated with 10 μg/mL AXL antibodies was tested asdescribed in Example 2. Pre-incubation with AXL antibody YW327.6S2, thatwas described to compete with Gas6 for AXL binding, IgG1-b12 (isotypecontrol) or medium (negative control) were included as controls.

Binding curves were analyzed using non-linear regression (sigmoidaldose-response with variable slope) using GraphPad Prism V5.04 software(GraphPad Software, San Diego, Calif., USA).

FIG. 12 and Table 11 shows that binding of Gas6 to A431 cells that hadbeen pre-incubated with IgG1-AXL-107 and IgG1-AXL-613 antibodies wassimilar to the IgG1-b12 and medium controls. This illustrates thatbinding of IgG1-AXL-107 and IgG1-AXL-613 to AXL does not interfere withGas6 binding, as shown in Example 2. The binding of Gas6 to A431 cellswas largely reduced in the presence of IgG1-AXL-061, IgG1-AXL-137 andcontrol AXL antibody YW327.652 compared to the IgG1-b12 and mediumcontrols.

In experiments in which A431 cells were pre-incubated with Gas6, themaximal binding values of IgG1-AXL-107 and IgG1-AXL-613 were comparableto antibody binding in absence of Gas6 (maximal binding after Gas6pre-incubation was 91-108% of binding without Gas6 pre-incubation)(Table 11). The EC₅₀ values for IgG1-AXL-107 and IgG1-AXL-613 bindingwith or without Gas6 pre-incubation were in the same range, or somewhathigher after Gas6 pre-incubation (Table 11), illustrating thatIgG1-AXL-107 and IgG1-AXL-613 do not compete with Gas6 binding.

Similar to control antibody YW327.6S2, the binding of IgG1-AXL-061 andIgG1-AXL-137 to A431 cells was greatly reduced in the presence of Gas6compared to binding without Gas6 (maximal binding after Gas6pre-incubation was 40-43% of binding without Gas6 pre-incubation; Table11). The EC₅₀ values for IgG1-AXL-061 and IgG1-AXL-137 could notproperly be determined after Gas6 pre-incubation (Table 11). This showsthat IgG1-AXL-061 and IgG1-AXL-137 compete with Gas6 for binding to AXL.

These data demonstrate that antibodies binding to the AXL Ig1 domainhave differential effect on Gas6 binding.

TABLE 11 Antibody binding to A431 cells Gas6 binding to A431 cellsMaximal Maximal binding in binding in presence presence of of Gas6 EC50in AXL antibodies EC50 w/o EC50 in (% of presence (% of binding Gas6EC50 presence binding in of AXL in presence (μg/mL) of Gas6 absence ofantibodies of control mean (μg/mL) Gas6) (μg/mL) antibody) Antibody(s.d.) mean (s.d.) mean (s.d.) mean (s.d.) mean (s.d.) IgG1-AXL-061 0.15(n.a.) n.a.  43 (28) n.a.  22 (8) IgG1-AXL-107 0.16 (0.17) 0.94 (1.18) 91 (5) 0.78 (0.54)  96 (8) IgG1-AXL-137 0.11 (0.10) n.a.  40 (18) n.a 36 (4) IgG1-AXL-613 0.09 (0.09) 0.10 (0.10) 108 (22) 0.57 (0.36) 100(11) YW327.6S2 0.09 (0.09) 1.90 (1.04)*  41 (24) 5.53 (7.09)*  17 (10)b12 n.a.^(a) n.a. n.a. 0.40 (0.11) 100 ^(a)n.a., not applicable *EC50values less accurate due to low binding.

Example 13—In Vivo Anti-Tumor Efficacy of AXL-ADCs in Xenograft Modelswith and without Autocrine (Endogenous) Gas6 Production

Gas6 Production of A431 and LCLC-103H Tumor Cells

It was tested whether A431 cells and LCLC-103H cells produce Gas6.Therefore, cells were grown in complete culture medium for 3 days. Gas6levels in supernatant were determined using the Quantikine Human Gas6ELISA (R&D Systems, Minneapolis, Minn.) according to manufacturer'sinstructions. This assay uses the quantitative sandwich ELISA technique.A monoclonal Ab specific for human Gas6 has been pre-coated onto amicroplate. Standards and samples are pipetted into the wells and anyhuman Gas6 present is bound by the immobilized Ab. After washing awayany unbound substances, an enzyme-linked polyclonal Ab specific forhuman Gas6 is added to the wells. Following a wash to remove any unboundAb-enzym reagent, a substrate is added to the wells and color developsin proportion to the amount of human Gas6 bound in the initial step. Thecolor development is stopped and the intensity of the color is measured.

Cell culture medium conditioned by A431 cells was found to contain 2576ng/mL Gas6, while the concentration of Gas6 in medium conditioned byLCLC-103H cells was more than 20-fold less (Table 12).

TABLE 12 Gas6 production in tumor cell conditioned medium. Gas6 insupernatant Cell line (ng/mL) LCLC-103H 126 A431 2576Anti-Tumor Activity of AXL-ADCs In Vivo

The in vivo anti-tumor activity of IgG1-AXL-061-vcMMAE (Ig1 binder),IgG1-AXL-107-vcMMAE (Ig1-binder), IgG1-AXL-137-vcMMAE (Ig1-binder),IgG1-AXL-148-vcMMAE (Ig2-binder), IgG1-AXL-183-vcMMAE (FN1-binder), andIgG1-AXL-726-vcMMAE (FN2-binder) was determined in the A431 (epidermoidcarcinoma) tumor model, that produces high levels of Gas6, and theLCLC-103H (large cell lung carcinoma) tumor model, that produces lowlevels of Gas6.

Tumor induction was performed by subcutaneous injection of 5×10⁶ A431 orLCLC-103H tumor cells (both obtained from Leibniz-Institut—DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ)) in 200 μL PBSin the right flank of female SCID mice. Treatment was started 14-21 daysafter tumor cell inoculation, when the average tumor size was >100-200mm³ and distinct tumor growth was observed. Mice received a singleinjection or a total of 4 biweekly intraperitoneal injections withIgG1-AXL-vcMMAE ADCs or control antibody (unconjugated IgG1-b12), asindicated. Tumor volume was determined at least two times per week.Tumor volumes (mm³) were calculated from caliper (PLEXX) measurementsas: 0.52×(length)×(width)².

FIG. 13A shows that treatment of mice with 3 mg/kg IgG1-AXL-107-vcMMAE,IgG1-AXL-148-vcMMAE and IgG1-AXL-733-vcMMAE induced growth inhibition ofA431 tumors.

FIG. 13B shows that treatment of mice with 3 mg/kg IgG1-AXL-148-vcMMAE,IgG1-AXL-183-vcMMAE (FN1 binder) and IgG1-AXL-726-vcMMAE (FN2 binder)induced growth inhibition of A431 tumors. In contrast, clonesIgG1-AXL-061-vcMMAE and IgG1-AXL-137-vcMMAE did not show anti-tumoractivity in the A431 xenograft model.

FIG. 14A shows that treatment of mice with 3 mg/kg IgG1-AXL-061-vcMMAE,IgG1-AXL-137-vcMMAE, IgG1-AXL-148-vcMMAE, IgG1-AXL-183-vcMMAE andIgG1-AXL-726-vcMMAE induced tumor regression in the LCLC-103H xenograftmodel. Similarly, treatment of mice with 1 mg/kg IgG1-AXL-107-vcMMAE or1 mg/kg IgG1-AXL-613-vcMMAE induced regression of LCLC-103H tumors (FIG.14B).

In summary, all AXL-ADCs showed anti-tumor activity in the LCLC-103Hxenograft model that produces low levels of Gas6. In the A431 xenograftmodel, that produces high levels of Gas6, anti-tumor activity was onlyobserved for those AXL-ADCs that did not compete with the AXL ligandGas6.

Example 14—AXL Expression in Different Tumor Indications

Expression of AXL was evaluated in freshly cut paraffin embedded andformalin fixated (FFPE) tumor tissue micro arrays (TMA) comprisingtissue cores from patients with thyroid, esophageal, ovarian,pancreatic, lung, breast, cervical or endometrial cancer, or malignantmelanoma. TMAs were obtained from US BioMax.

FFPE tumor array slides were deparaffinized and subjected to antigenretrieval (pH 6) and endogenous peroxidase was exhausted by incubationwith 0.1% H2O2 in citrate/phosphate buffer. To detect AXL expression,the TMAs were incubated with rabbit-anti-AXL (Santa Cruz, cat nr:sc-20741) at a concentration of 1 μg/mL for 60 min (room temperature(RT)). To identify (tumor) cells of epithelial origin, TMAs wereincubated with rabbit-anti-cytokeratin (Abcam, cat. Nr. ab9377) at adilution of 1:50 for 60 min (RT). After a washing step, the TMAs wereincubated with peroxidase conjugated, anti-rabbit IgG dextran polymer(ImmunoLogic, cat no: DPVR55HRP) to detect binding of rabbit Anti-AXLand rabbit anti-cytokeratin antibodies. Finally, binding of anti-rabbitIgG dextran polymer was visualized with di-amino-benzadine (DAB; browncolor; DAKO, cat no: K346811). In the TMA with malignant melanoma tissuecores, binding of anti-rabbit IgG dextran polymer was visualized withamino-ethyl carbazole (AEC; red color; Vector, SK4200). Nuclei in TMAswere visualized with hematoxylin (blue color).

AXL and cytokeratin immunostained TMAs were digitized with an Aperioslide scanner at 20× magnification and immunostaining was quantifiedwith tissue image analysis software (Definiens Tissue Studio software,version 3.6.1), using a cell-based algorithm. The algorithm was designedto identify and quantify the percentage of AXL- or cytokeratin-positivecells in the biopsies (range 0-100%) and to quantify AXL stainingintensity in AXL-positive tumor cells (optical density (OD); range 0-3)in each tumor core. Tumor cells were scored AXL positive, when AXL ODwas at least 0.1. The percentage of AXL positive tumor cells per tumorcore (range 0-100%) was calculated by dividing the total number of AXLpositive cells by the total number of cytokeratin-positive cells insequential tumor cores. The average AXL staining intensity (OD) in eachtumor core was calculated by dividing the sum of AXL OD of all AXLpositive tumor cells by the number of AXL positive tumor cells.

Tumor array from patients with malignant melanoma were scored manually.AXL staining intensity was scored as either weak (1+), moderate (2+) orstrong (3+) and the percentage AXL positive melanoma cells was scored in10% intervals (range 0-100%).

FIG. 16 provides a graphical representation of AXL expression in tumorcores of thyroid, esophageal, ovarian, breast, lung, pancreatic,cervical and endometrial cancer. Table 13 shows the percentage of tumorcores that showed AXL expression in more than 10% of tumor cells, foreach indication. FIG. 17 shows a representative example of a tissue coreimmunostained for AXL, for each indication. The figures illustrateheterogeneous expression of AXL in the tumor issue.

TABLE 13 % tumor cores (patients) with >10% AXL- positive Tumorindication Subtype tumor cells Esophageal Adenocarcinoma (n = 19) 73cancer Squamous cell carcinoma (n = 60) 55 Ovarian cancer All subtypes(n = 52) 90 Pancreatic cancer All subtypes (n = 58) 60 Lung cancerSquamous cell carcinoma SSC (n = 52) 63 (NSCLC) Adenocarcinoma (n = 48)67 Lung cancer SCLC (n = 5) 60 (SCLC) Thyroid cancer All subtypes (n =48) 92 Uterine cancer All subtypes (n = 60) 88 Breast cancer TNBC (n =54) 24 Cervical cancer All subtypes (n = 54) 93 Melanoma Malignantmelanoma (n = 67)  6 Abbreviations used: NSCLC, non small cell lungcancer; SLCL, small cell lung cancer; TNBC, triple negative breastcancer

Example 15—AXL Antibodies Specifically Bind AXL but not Other TAMReceptor Family Members

Expression of Human AXL, MER, and TYRO3 in HEK-293F Cells

The following codon-optimized constructs for expression of variousfull-length proteins were generated: human (Homo sapiens) AXL (Genbankaccession no. NP_068713.2), human MER (Genbank accession no. EAW52096.1,and human TYRO3 (Genbank accession no. Q06418.1). The constructscontained suitable restriction sites for cloning and an optimal Kozak(GCCGCCACC) sequence [Kozak et al. (1999) Gene 234: 187-208]. Theconstructs were cloned in the mammalian expression vector pcDNA3.3(Invitrogen)

Freestyle™ 293-F (a HEK-293 subclone adapted to suspension growth andchemically defined Freestyle medium, (HEK-293F)) cells were obtainedfrom Invitrogen and transfected with the expression plasmids using293fectin (Invitrogen), according to the manufacturer's instructions andgrown for 24-48 hours.

Binding Study of AXL Antibodies to Human AXL, Human MER, or Human TYRO3

HEK-293F cells transiently transfected with expression constructs forfull length human AXL, MER, or TYRO3 were evaluated for binding ofHuMab-AXL antibodies by flow cytometry. Transfected HEK-293F cells wereincubated with serial dilutions of AXL-antibodies (4-fold dilutions;final concentration range 0.002-10 μg/mL) for 30 minutes at 4° C. Afterwashing three times in PBS/0.1% BSA/0.02% azide, cells were incubatedwith R-Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab′)2(Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.; cat. No.109-116-098) diluted 1/100 in PBS/0.1% BSA/0.02% azide (final volume 100μL). Next, cells were washed twice in PBS/0.1% BSA/0.02% azide,resuspended in 120 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACSCantoll (BD Biosciences). Staining with mouse anti-human Mer (R&DSystems, cat. Mab8912) and mouse anti-human Tyro3 (Dtk) (R&D Systems,cat. MAB859) were included as controls for expression, IgG1-b12 wasincluded as a non-binding isotype control antibody. Binding curves wereanalyzed using non-linear regression (sigmoidal dose-response withvariable slope) using GraphPad Prism V5.04 software (GraphPad Software,San Diego, Calif., USA).

FIG. 18A shows that Humab-AXL antibodies showed dose-dependent bindingto the HEK293 cells expressing human AXL. In contrast, no binding ofHuMab-AXL antibodies to cells expressing MER (FIG. 18B) or TYRO3 (FIG.18C) or to untransfected HEK293 cells (FIG. 18D) was observed. Stainingwith MER- and Tyro3-specific antibodies confirmed that transfected cellsshowed proper expression of MER (FIG. 18F) or TYRO3 (FIG. 18G),respectively.

Example 16—Internalization of Cell Surface Bound AXL Antibodies

Internalization of Cell Surface Bound HuMab-AXL Evaluated by FlowCytometry.

Internalization of cell surface bound HuMab-AXL antibodies to MDA-MB-231and Calu-1 cells (human lung carcinoma cell line; ATCC, catalognumberHTB-54) was evaluated by flow cytometry. 50,000 cells were seeded in96-well tissue culture plates and allowed to attach for 6 hrs at 37° C.Plates were incubated at 4° C. for 30 minutes before incubation withserial dilutions of AXL-antibodies (final concentration range 0.0032-10μg/mL) at 4° C. for 1 hour. Subsequently, the medium was replaced bytissue culture medium without antibody and cells were incubatedovernight (16-18 hours) at 37° C. or 4° C. Subsequently, the cells weredetached with 40 μL warm trypsin solution, washed with ice-cold PBS/0.1%BSA/0.02% azide, and incubated for 30 minutes at 4° C. withR-Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab′)2 (JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.; cat. No.109-116-098) diluted 1/100 in PBS/0.1% BSA/0.02% azide (final volume 100μL), to detect AXL-antibodies on the cell surface. Finally, cells werewashed twice in PBS/0.1% BSA/0.02% azide, resuspended in 120 μL PBS/0.1%BSA/0.02% azide and analyzed on a FACS Cantoll (BD Biosciences).

Binding curves were analyzed using non-linear regression (sigmoidaldose-response with variable slope) using GraphPad Prism V5.04 software(GraphPad Software, San Diego, Calif., USA).

FIG. 19 shows that, for all AXL HuMab antibodies and at allconcentrations tested, more antibody was detected on the plasma membraneof cells that had been incubated at 4° C. after antibody binding,compared to cells that had been incubated at 37° C. This illustratesthat, at 37° C., AXL antibodies are internalized upon binding to theplasma membrane.

Fab-TAMRA/QSY7 Internalization and Intracellular Degradation Assay

Internalization of AXL antibodies was assessed in the Fab-TAMRA/QSY7internalization assay. This assay uses a fluorophore (TAMRA) andquencher (QSY7) pair. In close proximity, for example, upon conjugationto the same protein, TAMRA fluorescence is quenched by QSY7. In thisexample, goat-anti-human IgG Fab-fragments (Jackson Immunoresearch) wereconjugated with TAMRA/QSY7 (Fab-TAMRA/QSY7) as described (Ogawa et al.Mol Pharm 2009; 6(2):386-395), and AXL HuMab (1.5 μg/mL) werepreincubated with Fab-TAMRA/QSY7 (12 μg/mL; 30 min, 4° C.). The complexwas subsequently added to LCLC-103H cells and incubated for 24 hincubation in the dark, under shaking conditions (200 rpm, 37° C.).Internalization of the HuMab-Fab-TAMRA/QSY7 complex and intracellulardegradation in the endosomes and lysosomes causes dissociation ofTAMRA/QSY7, resulting in dequenching of TAMRA. TAMRA fluorescence ofLCLC-103H cells that had been incubated with AXL antibodies complexedwith Fab-TAMRA/QSY7 was measured on a FACS Canto-II (BD Biosciences).

As shown in FIG. 20, the fluorescence intensity of LCLC-103H cells wasenhanced upon incubation with AXL-antibody-Fab-TAM RA/QSY7 complex,compared to IgG1-b12-Fab-TAMRA/QSY7 or Fab-TAMRA/QSY7 alone. Thisillustrates that AXL antibodies are internalized upon binding toLCLC-103H cells.

Example 17—Anti-Tumor Efficacy of AXL-ADCs in an Esophageal CancerPatient-Derived Xenograft (PDX) Model

The anti-tumor activity of IgG1-AXL-107-vcMMAE was evaluated in thesubcutaneous esophageal PDX model ES0195 in BALB/c nude mice(experiments performed by Crown Bioscience. Taicang Jiangsu Province,China). Tumor fragments from donor mice bearing patient-derivedesophageal xenografts (ES0195) were used for inoculation into BALB/cnude mice. Each mouse was inoculated subcutaneously at the right flankwith one tumor fragment (2-3 mm in diameter) and tumors were allowed togrow until the tumor volume was about 150 mm³. Randomization of animalswas performed as follows: animals bearing a tumor with a volume of about150 mm³ were distributed in 5 experimental groups (8 animals per group),considering a comparable median and mean of group tumor volume. Thetreatment groups were: IgG1-b12, IgG1-b12-vcMMAE, IgG1-AXL-107,IgG1-AXL-107-vcMMAE, and paclitaxel. The antibodies and ADCs were dosedintravenously (i.v.) at 4 mg/kg at day of randomization (day 0) and day7. Paclitaxel was dosed intra-peritoneally (i.p.) at 20 mg/kg at day 0,7, and 14. Tumor volumes (mm³) were monitored twice weekly and werecalculated from caliper (PLEXX) measurements as: 0.52×(length)×(width)2.

FIG. 21 shows that treatment of mice with IgG1-AXL-107-vcMMAE inducedtumor regression of ES0195 tumors compared to the IgG1-b12 andIgG1-b12-MMAE control groups (p<0.001 at day 23, one-way ANOVA test).Treatment of mice with the untargeted ADC IgG1-b12-vcMMAE did not showanti-tumor activity in this model, illustrating that the therapeuticcapacity of AXL-ADCs depends on specific target binding. Mice that weretreated with paclitaxel showed tumor growth inhibition, but this wasless effective compared to treatment with IgG1-AXL-107-vcMMAE (p<0.05 atday 23, one-way ANOVA test).

Example 18—Anti-Tumor Efficacy of AXL-ADCs in a Cervical CancerPatient-Derived Xenograft (PDX) Model

The anti-tumor activity of IgG1-AXL-183-vcMMAE and IgG1-AXL-726-vcMMAEwas evaluated in the patient derived cervix carcinoma xenograft CEXF 773model in NMRI nu/nu mice (Harlan, Netherlands). Experiments wereperformed by Oncotest (Freiburg, Germany).

Tumor fragments were obtained from xenografts in serial passage in nudemice. After removal from donor mice, tumors were cut into fragments (4-5mm diameter) and placed in PBS (with 10% penicillin/streptomycin) untilsubcutaneous implantation. Mice under isofluorane anesthesia receivedunilateral, subcutaneous tumor implants in the flank. Tumors wereallowed to grow until the tumor volume was 50-250 mm³.

Randomization of animals was performed as follows: animals bearing atumor with a volume of 50-250 mm³ were distributed in 4 experimentalgroups (8 animals per group), considering a comparable median and meanof group tumor volume. The treatment groups were: IgG1-b12,IgG1-b12-vcMMAE, IgG1-AXL-183-vcMMAE and IgG1-AXL-726-vcMMAE. Theantibodies and ADCs were dosed intravenously (i.v.) at 4 mg/kg on theday of randomization (day 0) and on day 7. Tumor volumes (mm³) weremonitored twice weekly and were calculated from caliper (PLEXX)measurements as: 0.52×(length)×(width)².

FIG. 22 shows that treatment of mice with IgG1-AXL-183-vcMMAE orIgG1-AXL-726-vcMMAE induced tumor regression of CEXF 773 tumors comparedto the IgG1-b12 and IgG1-b12-MMAE control groups. Treatment of mice withthe untargeted ADC IgG1-b12-vcMMAE did not show anti-tumor activity inthis model, illustrating that the therapeutic capacity of AXL-ADCsdepends on specific target binding. Statistical analysis of tumor sizeat day 28 (Kruskal-Wallis and Mantel-Cox using a tumor size cut-off 500mm³), showed that the average tumor size in mice treated withIgG1-AXL-183-vcMMAE or IgG1-AXL-726-vcMMAE was significantly smallerthan in mice that had been treated with IgG1-b12 and IgG1-b12-vcMMAE(p<0.001). IgG1-AXL-183-vcMMAE and IgG1-AXL-726-vcMMAE were equallyeffective.

Example 19—Anti-Tumor Efficacy of AXL-ADCs in an Orthotopic BreastCancer Xenograft Model

The anti-tumor activity of IgG1-AXL-183-vcMMAE and IgG1-AXL-726-vcMMAEwas evaluated in in an orthotopic MDA-MB-231 D3H2LN xenograft model.

MDA-MB-231-luc D3H2LN Bioware cells (mammary gland adenocarcinoma;Perkin Elmer, Waltham, Mass.) were implanted in the mammary fat pad of6-11 week old, female SCID (C.B-17/IcrPrkdc-scid/CRL) mice(Charles-River) under isofluorane anesthesia. Tumors were allowed togrow and mice were randomized when tumors reached a volume of ^(˜)325mm³. Therefore, mice were distributed in 4 experimental groups (6-7animals per group), considering a comparable median and mean of grouptumor volume. The treatment groups were: IgG1-b12, IgG1-b12-vcMMAE,IgG1-AXL-183-vcMMAE and IgG1-AXL-726-vcMMAE. The animals received atotal of 4 biweekly doses of 3 mg/kg antibody or ADC starting at the dayof randomization. Tumor volumes (mm³) were monitored twice weekly andwere calculated from caliper (PLEXX) measurements as:0.52×(length)×(width)².

FIG. 23 shows that treatment of mice with IgG1-AXL-183-vcMMAE orIgG1-AXL-726-vcMMAE induced tumor regression of MDA-MB-231 tumorscompared to the IgG1-b12 and IgG1-b12-MMAE control groups. Treatment ofmice with the untargeted ADC IgG1-b12-vcMMAE did not show anti-tumoractivity in this model, showing that the therapeutic capacity ofAXL-ADCs depends on specific target binding. Statistical analysis oftumor size at day 32 (One Way Anova test), showed that the average tumorsize in mice that had been treated with IgG1-AXL-183-vcMMAE orIgG1-AXL-726-vcMMAE was significantly smaller than in mice that had beentreated with IgG1-b12 and IgG1-b12-vcMMAE (P<0.001). No differences wereobserved between the IgG1-AXL-183-vcMMAE and IgG1-AXL-726-vcMMAEtreatment groups, illustrating that these induced equally effectiveanti-tumor activity.

Example 20—In Vitro Cytotoxicity Induced by AXL-Specific Antibody DrugConjugates is Dependent on Target Expression

The in vitro cytotoxicity of IgG1-AXL-107-vcMMAE was tested in humantumor cell lines with different levels of AXL expression.

Cell Culture

LS174T cells (human colorectal adenocarcinoma cell line; ATCC, cat noCL-188) were cultured in Minimum Essential Medium (MEM) with Glutamax,Hepes and Phenol Red (Life Technologies, cat no 42360-024). Componentsare 10% Donor Bovine Serium with Iron (DBSI) (Life Technologies, cat no10371-029) and 1% Sodium Pyruvate (100 mM; Lonza, cat no 13E13-115E) and1% Penicillin/Streptomycin (Lonza, cat no DE17-603E).

NCI-H226 cells (human lung squamous cell carcinoma; ATCC, cat noCRL-5826), NCI-H661 cells (human large cell lung cancer; ATCC, cat noHTB-183), and NCI-H1299 cells (human non-small cell lung cancer; ATCC,cat no CRL-5803) were cultured in RPMI 1640 Medium (ATCC Modification;Life Technologies, cat no A10491-01). Components are 10% Donor BovineSerium with Iron (DBSI; Life Technologies, cat no 10371-029) and 1%Penicillin/Streptomycin (Lonza, cat no DE17-603E).

SKOV-3 cells (human ovarian adenocarcinoma; ATCC, cat no HTB-77) werecultured in McCoy's 5A Medium with L-glutamine and HEPES (Lonza, cat noBE12-168F). Components are 10% Donor Bovine Serium with Iron (DBSI; LifeTechnologies, cat no 10371-029) and 1% Penicillin/Streptomycin (Lonza,cat no DE17-603E).

Calu-1 cells (human lung epidermoid carcinoma; ATCC, cat no HTB-54) werecultured in McCoy's 5A Medium with Catopeptone, without HEPES (LifeTechnologies, cat no 26600-023). Components are 10% Donor Bovine Seriumwith Iron (DBSI; Life Technologies, cat no 10371-029) and 1% L-glutamine(200 nM) in 0.85% NaCl solution (Lonza, cat no BE17-605F) and 1%Penicillin/Streptomycin (Lonza, cat no DE17-603E). Calu-1 cells areresistant to EGFR targeted therapy.

LCLC-103H cells (human large cell lung cancer), A431 cells (humanepidermoid adenocarcinoma) and MDA-MB-231 cells (human breast cancer)were cultured as described in Example 8.

Quantification of AXL Expression on the Plasma Membrane of Human TumorCell Lines

AXL expression on the plasma membrane of human tumor cell lines wasassessed by indirect immunofluorescence using QIFIKIT (DAKO, Cat nrK0078) with mouse monoclonal antibody Z49M (Santa Cruz biotechnology,Cat nr sc-73719). Adherent cells were trypsinized and passed through acell strainer to obtain single cell suspensions. Cells were pelleted bycentrifugation for 5 minutes at 1,200 rpm, washed with PBS andresuspended at a concentration of 1×10⁶ cells/mL. The next steps wereperformed on ice. 100 μL of the single cell suspensions (100,000 cellsper well) were seeded in polystyrene 96-well round-bottom plates(Greiner Bio-One, Cat nr 650101). Cells were pelleted by centrifugationfor 3 minutes at 300×g and resuspended in 50 μL antibody sample or mouseIgG1 isotype control sample (BD/Pharmingen, Cat nr 555746) at aconcentration of 10 μg/mL. After an incubation of 30 minutes at 4° C.,cells were pelleted and resuspended in 150 μL FACS buffer. Set-up andcalibration beads were added to the plate according to themanufacturer's instructions. Cells and beads in parallel were washed twomore times with 150 μL FACS buffer and resuspended in 50 μLFITC-conjugated goat-anti-mouse IgG (1/50; DAKO, Cat nr K0078).Secondary antibody was incubated for 30 minutes at 4° C. in the dark.Cells and beads were washed twice with 150 μL FACS buffer andresuspended in 100 μL FACS buffer. Immunofluorescence was measured on aFACS Canto II (BD Biosciences) by recording 10,000 events within thegate of viable cells. The mean fluorescence intensity of the calibrationbeads was used to calculate the calibration curve using GraphPad Prismsoftware (GraphPad Software, San Diego, Calif., USA). For each cellline, the antibody binding capacity (ABC), an estimate for the number ofAXL molecules expressed on the plasma membrane, was calculated using themean fluorescence intensity of the AXL antibody-stained cells, based onthe equation of the calibration curve (interpolation of unknowns fromthe standard curve, using GraphPad Software).

Cytotoxicity Assay

For LCLC-103H, A431, MDA-MB-231, NCI-H226, NCI-H661, NCI-H1299, LS174Tand SKOV-3 cells, the in vitro cytotoxicity assay was performed asdescribed in Example 8. For Calu-1, the cytotoxicity assay was performedas described in Example 8, with the adaptation that the cell cultureswere incubated for 11 instead of 5 days. Dose-response curves weregenerated using Graphpad Prism software, using non-linear regressionanalysis. The percentage of viable cells at an IgG1-AXL-107-vcMMAEconcentration of 1 μg/mL was interpolated from the dose-response curves.

As shown in FIG. 24, IgG1-AXL-107-vcMMAE induced the most potentcytotoxicity in cell lines with high AXL expression, whereascytotoxicity was low or absent in cell lines with low AXL expression.The figure also illustrates that IgG1-AXL-107-vcMMAE is effective ininduction of cytotoxicity in cells resistant to EGFR targeted therapy,such as Calu-1.

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The invention claimed is:
 1. An immunoconjugate comprising (a) anantibody which binds to AXL (SEQ ID NO: 130) and comprises a heavy chainvariable (VH) region and a light chain variable (VL) region, wherein theVH region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQID NOs: 36, 37, and 38, respectively, and the VL region comprises theCDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 39, the aminoacid sequence GAS, and SEQ ID NO: 40, respectively, (b) a mc-vc-PABlinker, and (c) a cytotoxic agent, which is MMAE.
 2. The immunoconjugateof claim 1, wherein the VH and VL regions comprise the amino acidsequences of SEQ ID NOs: 1 and 2, respectively.
 3. The immunoconjugateof claim 1, wherein MMAE is linked to the antibody via the mc-vc-PABlinker, wherein the cytotoxic agent and the linker have the chemicalstructure;

wherein MAb is the antibody.
 4. The immunoconjugate according to claim1, wherein the MMAE is conjugated to the antibody via a cysteine thiolor a lysine.
 5. The immunoconjugate of claim 1, wherein cytotoxic agentloading (p) is 1 to
 8. 6. The immunoconjugate of claim 1, whereincytotoxic agent loading (p) is 10 to
 30. 7. The immunoconjugate of claim1, wherein said antibody comprises a heavy chain of an isotype selectedfrom the group consisting of IgG1, IgG2, IgG3, and IgG4.
 8. Theimmunoconjugate of claim 1, wherein the antibody is a full-lengthmonoclonal antibody.
 9. The immunoconjugate of claim 1, wherein theantibody is a full-length monoclonal IgG1,κ antibody.
 10. Theimmunoconjugate of claim 1, wherein the antibody is a monovalentantibody.
 11. The immunoconjugate of claim 1, wherein the antibody is anantigen-binding fragment of an immunoglobulin molecule.
 12. Theimmunoconjugate of claim 1, wherein the antibody is a single-chainantibody.
 13. The immunoconjugate of claim 1, wherein the antibody is ahuman antibody.
 14. A pharmaceutical composition comprising theimmunoconjugate of claim 1, and a pharmaceutical acceptable carrier. 15.A kit comprising the immunoconjugate of claim
 1. 16. The immunoconjugateof claim 6, wherein cytotoxic agent loading (p) is 1 to
 20. 17. Theimmunoconjugate of claim 6, wherein cytotoxic agent loading (p) is 2 to6.
 18. The immunoconjugate of claim 6, wherein cytotoxic agent loading(p) is 2 to
 4. 19. An immunoconjugate comprising (a) an antibody whichbinds to AXL (SEQ ID NO: 130) and comprises a heavy chain variable (VH)region and a light chain variable (VL) region, wherein the VH regioncomprises the amino acid sequence of SEQ ID NO: 1 and the VL regioncomprises the amino acid sequence of SEQ ID NO: 2, (b) a mc-vc-PABlinker, and (c) a cytotoxic agent, which is MMAE.
 20. Theimmunoconjugate of claim 19, wherein the combination of the MMAE and themc-vc-PAB linker has the chemical structure;

wherein MAb is the antibody.
 21. The immunoconjugate according to claim19, wherein the MMAE is conjugated to the antibody via a cysteine thiolor a lysine.
 22. The immunoconjugate of claim 19, wherein cytotoxicagent loading (p) is 1 to
 8. 23. The immunoconjugate of claim 19,wherein cytotoxic agent loading (p) is 10 to
 30. 24. The immunoconjugateof claim 19, wherein said antibody comprises a heavy chain of an isotypeselected from the group consisting of IgG1, IgG2, IgG3, and IgG4. 25.The immunoconjugate of claim 19, wherein the antibody is a full-lengthmonoclonal antibody.
 26. The immunoconjugate of claim 19, wherein theantibody is a full-length monoclonal IgG1,κ antibody.
 27. Theimmunoconjugate of claim 19, wherein the antibody is a monovalentantibody.
 28. The immunoconjugate of claim 19, wherein the antibody is asingle-chain antibody.
 29. The immunoconjugate of claim 19, wherein theantibody is an antigen-binding fragment of an immunoglobulin molecule.30. The immunoconjugate of claim 19, wherein the antibody is a humanantibody.
 31. A pharmaceutical composition comprising the antibody ofclaim 19, and a pharmaceutical acceptable carrier.
 32. A kit comprisingthe immunoconjugate of claim
 19. 33. The immunoconjugate of claim 19,wherein cytotoxic agent loading (p) is 1 to
 20. 34. The immunoconjugateof claim 19, wherein cytotoxic agent loading (p) is 2 to
 6. 35. Theimmunoconjugate of claim 19, wherein cytotoxic agent loading (p) is 2 to4.